Substituted amino acids as erythropoietin mimetics

ABSTRACT

This invention relates to a series of substitituted amino acids of Formula I 
                         
pharmaceutical compositions containing them and intermediates used in their manufacture. The compounds of the invention are small molecules which bind to the erythropoietin receptor and compete with the natural ligand for binding to this receptor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 10/799,324, now Pat. No.7,153,869, filed Mar. 12, 2004, which is a divisional of Ser. No.09/927,111, now Pat. No. 6,750,369, filed Aug. 10, 2001, which is adivisional of Ser. No. 09/517,976, now Pat. No. 6,310,078, filed Mar. 3,2000, which is a divisional of Ser. No. 09/294,785, now abandoned, filedApr. 19, 1999, which claims priority from a Provisional Ser. No.60/082,392, filed Apr. 20, 1998. The complete disclosures of theaforementioned applications are incorporated herein by reference intheir entirety.

This invention relates to a series of small molecules which bind to theerythropoietin receptor and compete with the natural ligand for bindingto said receptor. The invention includes pharmaceutical compositionscontaining these mimetics, their methods of production as well asintermediates used in their synthesis.

Erythropoietin (EPO) is a 34,000 dalton glycoprotein hormone which isproduced in the mammalian kidney. Its primary role is stimulation ofmitotic cell division and differentiation of erythrocyte precursorcells. As a result this hormone regulates the production oferythrocytes, the hemoglobin contained therein and the blood's abilityto carry oxygen. The commercial product Epogen® is used in the treatmentof anemia. This drug is produced by recombinant techniques and isformulated in aqueous isotonic sodium chloride/sodium citrate. Eventhough it has been, used successfully in the treatment of anemia, it isa costly drug that is administered intravenously. This method ofadministration is both costly and inconvenient for the patient;therefore it would be desirable to find a EPO mimetic which has thepotential for oral activity.

A small molecule EPO mimetic has advantages over the natural protein.The immune response associated with large peptides is unlikely to occurwith small molecules. In addition, the variety of pharmaceuticalformulations that may be used with small molecules are technicallyunfeasible for proteins. Thus the use of relatively inert formulationsfor small molecules is possible. The most important advantage of smallmolecules is their potential for oral activity. Such an agent would easeadministration, cost less and facilitate patient compliance.

Although compounds which mimic EPO are useful in stimulating red bloodcell synthesis, there are diseases where the overproduction of red bloodcells is a problem. Erythroleukemia and polysythemia vera are examplesof such diseases. Since EPO is an agent responsible for the maturationof red blood cell precursors, an antagonist of EPO would have utilitytreating either of those diseases.

SUMMARY OF THE INVENTION

The disclosed invention consists of a series of small molecules whichdemonstrate competitive binding with the natural ligand for the EPOreceptor. As such these compounds are potentially useful in thetreatment of diseases or conditions associated with this receptor. Inaddition, the invention contemplates methods of producing thesecompounds and intermediates used in their production.

The invention includes compounds of the Formula I:

wherein:

-   -   R¹ is the side chain of a natural or unnatural α-amino acids,        where if said side chain contains a protectable group, that        group may be protected with a member of the group consisting of        succinyl, glutaryl, 3,3-dimethylglutaryl, C₁₋₅alkyl,        C₁₋₅alkoxycarbonyl, acetyl, N-(9-fluorenylmethoxycarbonyl),        trifluoroacetyl, omega-carboxyC₁₋₅alkylcarbonyl,        t-butoxycarbonyl, benzyl, benzyloxycarbonyl,        2-chlorobenzyloxycarbonyl, phenylsulfonyl, ureido, t-butyl,        cinnamoyl, trityl, 4-methyltrityl,        1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,        4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and        substituted phenylureido (where the phenyl substituents are        phenoxy, halo, C₁₋₅alkoxycarbonyl);    -   R² and R³        -   may be taken together to form a six-membered aromatic ring            which is fused to the depicted ring, or        -   are independently selected from the group consisting of            hydrogen, C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, amino, phenyl, phenoxy,            phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy,        -   substituted phenyl (where the substituents are selected from            C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,            nitro, cyano, and is amino),        -   substituted phenoxy (where the substituents are selected            from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, cyano, and amino),        -   substituted phenylC₁₋₅alkyl (where the substituents are            selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, cyano, and amino),        -   substituted phenylC₁₋₅alkoxy (where the substituents are            selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, cyano, and amino), and        -   substituted amino (where the substituents are selected from            one or more members of the group consisting of C₁₋₅alkyl,            halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,            phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted            C₁₋₅alkylcarbonyl, carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl,            cinnamoyl, naphthylcarbonyl, furylcarbonyl, pyridylcarbonyl,            C₁₋₅alkylsulfonyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,            phenylsulfonyl, phenylC₁₋₅alkylsulfonyl substituted            phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,            substituted phenylsulfonyl, substituted            phenylC₁₋₅alkylsulfonyl, substituted phenyl, and substituted            phenylC₁₋₅alkyl [where the aromatic phenyl, phenylC₁₋₅alkyl,            phenylcarbonyl, phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, and            phenylC₁₋₅alkylsulfonyl substitutents are independently            selected from one to five members of the group consisting of            C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl,            nitro, cyano, and amino]);    -   R⁴ and R⁵        -   may be taken together to form a six-membered aromatic ring            which is fused to the depicted ring, or        -   are independently selected from the group consisting of            hydrogen, C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, amino, phenyl, phenoxy,            phenylC₁₋₅alkyl, phenyl C₁₋₅alkoxy,        -   substituted phenyl (where the substituents are selected from            C₁₋₅alkyl, C₁₋₅ alkoxy, hydroxy, halo, trifluoromethyl,            nitro, cyano, and amino),        -   substituted phenoxy (where the substituents are selected            from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, cyano, and amino),        -   substituted phenylC₁₋₅alkyl (where the substituents are            selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, cyano, and amino),        -   substituted phenylC₁₋₅alkoxy (where the substituents are            selected from C₁₋₅ alkyl, C₁₋₅ alkoxy, hydroxy, halo,            trifluoromethyl, nitro, cyano, and amino), and        -   substituted amino (where the substituents are selected from            one or more members of the group consisting of C₁₋₅alkyl,            halosubstitutedC₁₋₅alkyl, C₁₋₅alknyl, C₁₋₅alkenyl, phenyl,            phenylC₁₋₅alkyl, C₁₋₅alkylcarbonyl, halo substituted            C₁₋₅alkylcarbonyl, carboxyC₁₋₅alkyl, C₁₋₅alkoxyC₁₋₅alkyl,            cinnamoyl, naphthylcarbonyl, furylcarbonyl, pyridylcarbonyl,            C₁₋₅alkylsulfonyl, phenylcarbonyl, phenylC₁₋₅alkylcarbonyl,            phenylsulfonyl, phenylC₁₋₅alkylsulfonyl substituted            phenylcarbonyl, substituted phenylC₁₋₅alkylcarbonyl,            substituted phenylsulfonyl, substituted            phenylC₁₋₅alkylsulfonyl, substituted phenyl, and substituted            phenylC₁₋₅alkyl [where the aromatic phenyl, phenylC₁₋₅alkyl,            phenylcarbonyl, phenylC₁₋₅alkylcarbonyl, phenylsulfonyl, and            phenylC₁₋₅alkylsulfonyl substitutents are independently            selected from one to five members of the group consisting of            C₁₋₅alkyl, C₁₋₅alkoxy, hydroxy, halogen, trifluoromethyl,            nitro, cyano, and amino]);    -   W is selected from the group consisting of —CH═CH—, —S—, and        —CH═N—;    -   Q is selected from the group consisting of —CH═CH—, —S—, and        —CH═N—;    -   X is selected from the group consisting of carbonyl, C₁₋₅alkyl,        C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is        2-5;    -   Y is selected from the group consisting of carbonyl, C₁₋₅alkyl,        C₁₋₅alkenyl, C₁₋₅alkenylcarbonyl, and (CH₂)_(m)—C(O)— where m is        2-5;    -   n is 1, 2, or 3;    -   Z is selected from the group consisting of hydroxy, C₁₋₅ alkoxy,        phenoxy, phenylC₁₋₅alkoxy, amino, C₁₋₅alkylamino,        diC₁₋₅alkylamino, phenylamino, phenylC₁₋₅alkylamino,        piperidin-1-yl    -   substituted piperidin-1-yl (where the substituents are selected        from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy, halo,        aminocarbonyl, C₁₋₅alkoxycarbonyl, and oxo;    -   substituted phenylC₁₋₅alkylamino (where the aromatic        substitutents are selected from the group consisting of        C₁₋₅alkyl, C₁₋₅alkoxy, phenylC₁₋₅alkenyloxy, hydroxy, halogen,        trifluoromethyl, nitro, cyano, and amino),    -   substituted phenoxy (where the aromatic substitutents are        selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy,        hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),    -   substituted phenylC₁₋₅alkoxy (where the aromatic substitutents        are selected from the group consisting of C₁₋₅alkyl, C₁₋₅alkoxy,        hydroxy, halogen, trifluoromethyl, nitro, cyano, and amino),    -   —OCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂O—,    -   —NHCH₂CH₂(OCH₂CH₂)_(s)OCH₂CH₂NH—,    -   —NH(CH₂)_(p)O(CH₂)_(q)O(CH₂)_(p)NH—,        —NH(CH₂)_(q)NCH₃(CH₂)_(s)NH—,    -   —NH(CH₂)_(s)NH—, and (NH(CH₂)_(s))₃N,        -   where s, p, and q are independently selected from 1-7    -   with the proviso that if n is 2, Z is not hydroxy, C₁₋₅ alkoxy,        amino, C₁₋₅alkylamino, diC₁₋₅alkylamino, phenylamino,        phenylC₁₋₅alkylamino, or piperidin-1-yl,        -   with the further proviso that if n is 3, Z is            (NH(CH₂)_(s))₃N.            and the salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The terms used in describing the invention are commonly used and knownto those skilled in the art. “Independently” means that when there aremore than one substituent, the substitutents may be different. The term“alkyl” refers to straight, cyclic and branched-chain alkyl groups and“alkoxy” refers O-alkyl where alkyl is as defined supra. “Cbz” refers tobenzyloxycarbonyl. “Boc” refers to t-butoxycarbonyl and “Ts” refers totoluenesulfonyl. “DCC” refers to 1,3-dicyclohexylcarbodiimide, “DMAP”refers to 4-N′,N-dimethylaminopyridine and “HOBT” refers to1-hydroxybenzotriazole hydrate. “Fmoc” refers toN-(9-fluorenylmethoxycarbonyl), “DABCO” refers to1,4-Diazabicyclo[2.2.2]octane, “EDCI” refers to1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, “Dde” refers to1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, and “TMOF” refers totrimethyl orthoformate. The side chains of α-amino acids refer to thesubstituents of the stereogenic carbon of an α-amino acid. For exampleif the amino acid is lysine, the side chain is 1-aminobutan-4-yl. Theterm natural amino acid refers to the 20 α-amino acids of the Lconfiguration which are found in natural proteins. Unnatural α-aminoacids include synthetic amino acids such as, α-aminoadipic acid,4-aminobutanoic acid, 6-aminohexanoic acid, α-aminosuberic acid,5-aminopentanoic acid, p-aminophenylalanine, α-aminopimelic acidγ-carboxyglutamic acid, p-carboxyphenylalanine, carnitine, citrulline,α,β-diaminopropionic acid, α,γ-diaminobutyric acid, homocitrulline,homoserine, and statine as well as D-configuration amino acids. The term“protectable group” refers to a hydroxy, amino, carboxy, carboxamide,guanidine, amidine or a thiol groups on an amino acid side. Compounds ofthe invention may be prepared by following general procedures known tothose skilled in the art, and those set forth herein.

The compounds of the invention may be prepared by liquid phase organicsynthesis techniques or by using amino acids which are bound to a numberof known resins. The underlying chemistry, namely, acylation andalkylation reactions, peptide protection and deprotection reactions aswell as peptide coupling reactions use similar conditions and reagents.The main distinction between the two methods is in the startingmaterials. While the starting materials for the liquid phase synthesesare the N-protected amino acids or the lower alkyl ester derivatives ofeither the N-protected or N-unprotected amino acids, the startingmaterial for the resin syntheses are N-protected amino acids which arebound to resins by their carboxy termini.

General Procedure for the Solid-Phase Synthesis of SymmetricalNα,Nα-Disubstituted Amino Acids Scheme 1

An equivalent of an N-Fmoc-protected amino acid which is bound to aresin 1a is suspended in a suitable solvent such as DMF. This solvent isremoved and the nitrogen protecting group (Fmoc) is removed by stirringthe resin bound amino acid with an organic base, such as piperidine, andan addition portion of the solvent. A solution of about two to threeequivalents of an appropriately substituted halide, 1b, and a suitablebase such DIEA is added to the resin bound amino acid and this mixtureis shaken for 18-36 h. The resulting mixture is washed with severalportions of a suitable solvent and is suspended and shaken in an acidicsolution, such as 50% TFA/CH₂Cl₂, over several hours to cleave the acidfrom the resin and give the N-disubstituted amino acid 1c.

By varying the resin bound amino acid 1a, one may obtain many of thecompounds of the invention. The following resin bound amino acids may beused in Scheme I: alanine,N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine,β-(4-methyltrityl)asparagine, aspartic acid (β-t-butyl ester),S-(trityl)cysteine, γ-(4-methyltrityl)glutamine, glutamic acid(β-t-butyl ester), glycine, N-imidazolyl-(trityl)histidine, isoleucine,leucine, N-ε-(2-chlorobenzyloxycarbonyl)lysine,N-ε-(t-butoxycarbonyl)lysine, methionine, phenylalanine, proline,O-(t-butyl)serine, O-(t-butyl)threonine,N-indolyl-(t-butoxycarbonyl)tryptophan. O-(t-butyl)tyrosine, valine,β-alanine, α-aminoadipic acid, 4-aminobutanoic acid, 6-aminohexanoicacid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine,α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine,carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyricacid, homocitrulline, homoserine, and statine. In addition, the choiceof “W” and “X” can be varied by using known halide derivatives of 1b.For example using benzylchloride, 2-chloromethylthiophene, or2-chloromethylpyridine gives compounds of the invention where “W” is—CH═CH—, —S—, or —CH═N—, respectively. For variations in “X”, the use of2-chloroethylphenyl, 3-chloro-1-propenylbenzene, or benzeneacetylchloride as 1b, give compounds where Y is (CH₂)₂, —CH═CH—CH₂—, or—CH₂C(O)— respectively. Still further, Scheme 1 may be used to producecombinatorial mixtures of products. Using mixtures of resin bound aminoacids, 1a, with only one 1b produces said combinatorial mixtures.Alternatively, using one amino acid 1a with a mixture of 1b as well asmixture of 1a with mixtures of 1b gives a large range of combinatorialmixtures.

General Procedure for the Solid-Phase Synthesis of UnsymmetricalNα,Nα-Disubstituted Amino Acids Scheme 2, Step A

An equivalent of an N-Fmoc-protected amino acid which is bound to aresin 1a is suspended in a suitable solvent such as DMF. This solvent isremoved and the nitrogen protecting group (Fmoc) is removed by stirringthe resin bound amino acid with an organic base, such as piperidine, andan addition portion of the solvent. Trimethyl orthoformate and anappropriately substituted aldehyde 2a (5 equivalents) is added and themixture is shaken under N₂ overnight. This mixture is treated with asuspension of NaBH(OAc)₃ (5 equivalents) in CH₂Cl₂ and shaken under N₂overnight. After filtration and washing with a suitable solvent, theresulting product, resin bound Nα-monosubstituted amino acid 2b, isrinsed with a suitable solvent and its identity is confirmed by MS andor HPLC analysis after treatmet of a portion of the resin with 50%TFA/CH₂Cl₂.

Scheme 2, Step B

The resin 2b is suspended in an appropriate solvent such as DMF and isfiltered. The appropriately substituted alkyl or arylkyl halide, 2c, andan appropriate base such as DIEA are added with some additional solventand the mixture is shaken under N₂ for 18-36 h. The resin boundNα,Nα-disubstituted amino acid, 2d, is isolated from the suspension andthe resin is cleaved with an acidic solution to give the free acid 2e.

Scheme 3, Step C

A resin bound amine, 2d, where R⁴ is nitro, is suspended in a suitablesolvent, such as DMF, and is filtered. This mixture is treated withSnCl₂ dihydrate in DMF and shaken under N₂ overnight. The solvent isremoved and the resin is washed successive portions of a suitablesolvent to give the resin bound compound 3a where R⁴ is amino. The resinis suspended in a suitable solvent and is combined with an organic base,such as pyridine an appropriately substituted carboxylic acid anhydride,acid chloride, or sulfonyl chloride. The mixture is shaken under N₂overnight and is filtered to give the resin bound amino acid 3b. Thismaterial is treated with an acid and a suitable solvent to give the freeamino acid 3b.

Scheme 3, Step D

The resin bound amine 3a is treated with TMOF and an appropriatelysubstituted aldehyde 3c is added and the mixture is shaken under N₂overnight. The resulting mixture is drained and treated with asuspension of NaBH(OAc)₃ in an appropriate solvent and this mixture isshaken under N₂ overnight. The resin bound 3-aralkylaminophenyl aminoacid is identified my spectral techniques after clevage to give the freeacid 3d as previously described.

Scheme 3, Step E

Resin bound, 2d, where R¹ is (CH₂)₄NH(Dde) is mixed with a suitablesolvent, such as DMF, and shaken with successive portions of 2% solutionof hydrazine hydrate in DMF over about 30 min. The resin is filtered andtreated with a suitable solvent and a cyclic anhydride derivative 3e,and a base such as DMAP and pyridine. This mixture is shaken under N₂overnight and filtered to give the resin bound amine, 3f. This materialis identified by spectral techniques after clevage to give the free acid3f as previously described.

Scheme 4, Step F

Resin bound 2b, where R² is nitro is suspended in CH₂Cl₂ and is treatedwith an organic base, such as pyridine, and 9-fluorenylmethoxy chloride.This mixture is shaken under N₂ overnight, filtered and resuspended in asuitable solvent. This mixture is treated with SnCl₂ dihydrate in DMFand shaken under N₂ overnight. The solvent is removed and the resin iswashed successive portions of a suitable solvent and filtered to givethe resin bound compound 4a where R² is amino. The resin 4a is thensuspended in a suitable solvent, such as CH₂Cl₂, and is combined with0.4 mmol of pyridine and 0.25-0.4 mmol of the appropriately substitutedcarboxylic acid anhydride, acid chloride, or sulfonyl chloride. Themixture is shaken under N₂ overnight, filtered, and washed successivelywith three portions each of CH₂Cl₂ and MeOH. This resin is suspended inDMF, filtered, and shaken under N₂ with 5 mL of a 40% solution ofpiperidine in DMF. After 1 h, the solvent is drained and the resin waswashed successively with three portions each of suitable solvents togive the resin bound 4b. The identity of the compound was confirmed byspectral analysis after cleveage as previously described.

Scheme 5

The resin 2b (0.2 mmol) is suspended in CH₂Cl₂, filtered, and isresuspended in CH₂Cl₂. This suspension is treated with diethylphosphonoacetic acid and diisopropylcarbodiimide or other suitablecarbodiimide reagent, and the mixture is shaken under N₂ overnight. Thesolvent is drained and the resulting resin 5a was washed successivelywith three portions each of CH₂Cl₂ and MeOH. The resin is suspended inDMF and filtered. A solution of the appropriately substituted aldehyde5b (0.6-1.0 mmol) in 3-5 mL of DMF, lithium bromide (0.6-1.0 mmol), anda suitable base such as DIEA or Et₃N (0.6-1.0 mmol) is added and themixture is shaken under N₂ overnight. The solvent is removed and theresin is washed successively with three portions each of DMF, CH₂Cl₂,and MeOH. The identity of the resin bound substituted amino acid 5c wasconfirmed spectral techniques. The resin bound material may be treatedwith 50% TFA/CH₂Cl₂ over 1-1.5 h, to give the acid 5c.

Scheme 6

To prepare compounds where n is 2 and Z is NH(CH₂)_(s)NH, products ofSchemes 1-5 may be used in Scheme 6. Treatment of two equivalents of thesubstituted amino acid 1c with an equivalent of the diamine 6a, in thepresence of HOBT and a peptide coupling agent such as EDCI and a basesuch as DIEA at room temperature over 16 h gives the dimer 6b.

General Procedure for the Solution-Phase Synthesis of SymmetricalNα,Nα-Disubstituted Amino Acids Scheme 7, Step A

A solution of of amino acid ester 7a, an appropriately substitutedhalide derivitive 1b, and an appropriate base such as DIEA, Na₂CO₃, orCs₂CO₃ in a suitable solvent, such as DMF, is heated at 50-100° C. underN₂overnight, or until the starting material is exhausted, to give amixture of the di and mono-substituted amines, 7b and 7c respectively.If the side chains of R¹ contain acid cleavable protecting groups, thosegroups may be cleaved by treatment with 30-80% TFA/CH₂Cl₂. Esters 7b and7c may be independently converted to the corresponding acids 7d and 7eby hydrolysis with an appropriate base such as aqueous NaOH.

General Procedure for the Solution-Phase Synthesis of UnsymmetricalNα,Nα-Disubstituted Amino Acids Scheme 8, Step A

A solution of 1 mmol of amino acid ester 8a (or the corresponding HClsalt and 1.1 mmol of DIEA) and 1-1.5 mmol of the appropriatelysubstituted aldehyde 2a in 3-5 mL of trimethyl orthoformate was stirredat room temperature under N₂ overnight. The solution was eitherconcentrated and used directly for the next reaction, or was partitionedbetween EtOAc and water, washed with brine, dried over Na₂SO₄, andconcentrated to give crude product, which was purified by MPLC to givemono-substituted product 8b.

Scheme 8, Step B

Amino ester 8b was dissolved in DMF, combined with 1.1-1.5 mmol of theappropriately substituted chloride or bromide 2c, and heated at 50-100°C. overnight. The reaction mixture was cooled and partitioned betweenwater and EtOAc. The organic layer was washed three times with water andonce with brine, dried over Na₂SO₄, and concentrated. The crude productwas purified by MPLC to give pure 8c. For examples of 8c wherein theside chain R¹ contained an acid-cleavable protecting group such ast-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was stirred in30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated andoptionally dissolved in HOAc and freeze-dried to give the deprotectedform of 8c. For examples of 8c where R⁹ was equal to t-butyl, 8c wasstirred in 30-80% TFA/CH₂Cl₂ for 1-3 h and treated as described above togive acid 8d. For examples of 8c where R⁹ was equal to methyl, ethyl, orother primary or secondary alkyl esters, 8c was stirred with with 1-2mmol of aqueous LiOH, NaOH, or KOH in MeOH, EtOH, or THF at 20-80° C.until TLC indicated the absence of 8c. The solution was acidified to pH4-5 with aqueous citric acid or HCl and was extracted with CH₂Cl₂ orEtOAc. The organic solution was washed with brine, dried over Na₂SO₄,and concentrated to give 8d.

Scheme 8, Step C

For examples of amino acid ester 8c where R¹=(CH₂)₄NHBoc, 8c (1 mmol)was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture wasconcentrated to provide 8e as the TFA salt. Optionally, the TFA salt wasdissolved in CH₂Cl₂ or EtOAc and washed with aqueous NaOH or Na₂CO₃,dried over Na₂SO₄, and concentrated to give 8e as the free base.

Scheme 8, Step D

A solution of 1 mmol of 8e, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride 3ewas stirred in CH₂Cl₂ or DMF under N₂ overnight. The resulting mixturewas diluted with CH₂Cl₂ or EtOAc and washed with aqueous HCl, water, andbrine, was dried over Na₂SO₄, and concentrated to provide 8f.Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted carboxylic acidanhydride (R¹¹CO)₂O or acid chloride R¹¹COCl was stirred in CH₂Cl₂ orDMF under N₂ overnight and worked up as above to provide 8g.Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted isocyanate R¹²NCOwas stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as aboveto provide 8h.

Scheme 9, Step A

For examples of 8c where R⁵═NO₂, a solution of 1 mmol of 8c (where R²,R³, R⁴, or) and 10-12 mmol of SnCl₂ dihydrate was stirred in MeOH, EtOH,or DMF at 20-80° C. for 0.5-24 h under N₂. The solution was taken toroom temperature and poured into aqueous Na₂CO₃ with rapid stirring. Theresulting mixture was extracted with EtOAc or CH₂Cl₂ and the organicextracts were washed with brine, dried over Na₂SO₄, and concentrated togive the aminophenyl product 9a, which was purified by MPLC or usedwithout further purification.

Scheme 9, Step B

A solution of 1 mmol of aminophenyl compound 9a and 1-1.5 mmol of theappropriately substituted aldehyde 2a in 3-5 mL of trimethylorthoformate was stirred at room temperature under N₂ overnight. Thesolution was either concentrated and used directly for the nextreaction, or was partitioned between EtOAc and water, washed with brine,dried over Na₂SO₄, and concentrated to give crude product, which waspurified by MPLC to give 9b. For examples of 9b wherein the side chainR¹ or R⁹ contained an acid-cleavable protecting group such ast-butylcarbamate, t-butyl ester, or t-butyl ether, 9b was stirred in30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture was concentrated andoptionally dissolved in HOAc and freeze-dried to give the deprotectedform of 9b.

Scheme 9, Step C

A solution of 1 mmol of 3-aminophenyl compound 9a, 1.1-2 mmol ofpyridine, and 1-1.5 mmol of the appropriately substituted acid chloride,acid anhydride, or sulfonyl chloride in 3-5 mL of CH₂Cl₂ or ClCH₂CH₂Clwas stirred at room temperature under N₂ overnight. The solution waspartitioned between EtOAc and water, washed with water, saturatedaqueous NaHCO₃, and brine, dried over Na₂SO₄, and concentrated to givecrude product which was optionally purified by MPLC to give amide orsulfonamide 9c. For examples of 9c wherein the side chain R¹ or R⁹contained an acid-cleavable protecting group such as t-butylcarbamate,t-butyl ester, or t-butyl ether, 9c was stirred in 30-80% TFA/CH₂Cl₂ for1-3 h. The reaction mixture was concentrated and optionally dissolved inHOAc and freeze-dried to give the deprotected form of 9c.

General Procedure for the Solution-Phase Synthesis of SymmetricalNα,Nα-Disubstituted Amino Amides and their Dimers and Trimers Scheme 10,Step A

A solution of 1 mmol of N-Cbz-protected amino acid 10a and theappropriate amine (ZH, 1 mmol), diamine (ZH₂, 0.5 mmol), or triamine(ZH₃ 0.33 mmol), was treated with 1.1 mmol of HOBt, 1.1 mmol of DIEA,and 2.1 mmol of EDCI in 3-6 mL of CH₂Cl₂ or DMF. [Alternatively, 1 mmolof the pentafluorophenyl ester or N-hydroxysuccinimide ester of 10a wasmixed with the appropriate portion of amine (ZH), diamine (ZH₂), ortriamine (ZH₃) in 3-6 mL of DMF.] The solution was stirred at roomtemperature under N₂ for 12-24 h, and EtOAc was added. The organicsolution was washed with 5% aqueous citric acid, water, saturatedNaHCO₃, and brine, dried over Na₂SO₄, and concentrated. The crudeproduct was optionally purified by MPLC to afford amide 10b. Compound10b was stirred in 30-80% TFA/CH₂Cl₂ for 1-3 h. The reaction mixture wasconcentrated to provide the TFA salt which was dissolved in CH₂Cl₂ orEtOAc and washed with aqueous NaOH or Na₂CO₃, dried over Na₂SO₄, andconcentrated to give 10c as the free base.

Scheme 10, Step B

A solution of 1 mmol of amino acid ester 10c (n=1), 2.5-3 mmol of theappropriately substituted chloride or bromide 2c, and 2.5-3 mmol of anappropriate base such as DIEA, Na₂CO₃, or Cs₂CO₃ in 3-5 mL of DMF washeated at 50-100° C. under N₂ for 18-24 h. (For examples of 10c wheren=2 or 3, the amounts of 2c and base were increased by two- orthree-fold, respectively.) The reaction mixture was cooled andpartitioned between water and EtOAc. The organic layer was washed threetimes with water and once with brine, dried over Na₂SO₄, andconcentrated. The crude product was purified by MPLC to give pure amide10d.

Alternatively, a solution of 1 mmol of amino acid ester 10c (n=1), 2.5-3mmol of the appropriately substituted aldehyde 2a, and 2.5-3 mmol ofborane-pyridine complex in 3-5 mL of DMF or EtOH was stirred at roomtemperature under N₂ for 3-5 days. (For examples of 10c where n=2 or 3,the amounts of 2c and borane-pyridine complex were increased by two- orthree-fold, respectively.) The mixture was concentrated to dryness andwas partitioned between water and CH₂Cl₂, washed with brine, dried overNa₂SO₄, and concentrated. The crude product was purified by MPLC to givepure amide 10d.

Scheme 10, Step C

For examples of 10d where R¹=CH₂CH₂CO₂-t-Bu or CH₂CO₂-t-Bu, 10d wasstirred in 30-80% TFA/CH₂Cl₂ for 1-24 h. The reaction mixture wasconcentrated and optionally dissolved in HOAc and freeze-dried to giveacid 10e.

Scheme 10, Step D

For examples of 10d where R¹ is equal to (CH₂)₄NHBoc, 10d was stirred in30-80% TFA/CH₂Cl₂ for 1-24 h. The reaction mixture was concentrated andoptionally dissolved in HOAc and freeze-dried to give amine 10f as theTFA salt which was optionally dissolved in CH₂Cl₂ or EtOAc, washed withaqueous NaOH or Na₂CO₃, dried over Na₂SO₄, and concentrated to give 10fas the free base.

Scheme 10, Step E

A solution of 1 mmol of 10f, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted cyclic anhydride 3ewas stirred in CH₂Cl₂ or DMF under N₂ overnight. The resulting mixturewas diluted with CH₂Cl₂ or EtOAc and washed with aqueous HCl, water, andbrine, was dried over Na₂SO₄, and concentrated to provide acid 10g.Alternatively, 1 mmol of 10f, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted carboxylic acidanhydride (R¹¹CO)₂O or acid chloride R¹¹COCl was stirred in CH₂Cl₂ orDMF under N₂ overnight and worked up as above to provide 10 h.Alternatively, 1 mmol of 8e, 1-4 mmol of an appropriate base such asDIEA, and 1-2 mmol of the appropriately substituted isocyanate R¹²NCOwas stirred in CH₂Cl₂ or DMF under N₂ overnight and worked up as aboveto provide 10i.

General Procedure for the Solid-Phase Synthesis Of Nα,Nα-Bis-CinnamylAmino Acids and Nα-Cinnamyl Amino Acids Scheme 11

An equivalent of an N-Fmoc-protected amino acid 11a which is bound to apolystyrene resin such as Wang resin is suspended in a suitable solventsuch as DMF. This solvent is removed and the nitrogen protecting group(Fmoc) is removed by stirring the resin bound amino acid with an organicbase, such as piperidine, and an addition portion of the solvent. Afterfiltration and washing with solvent, the resin is suspended in anappropriate solvent such as DMF. A solution of about 2-3 equivalents ofan appropriately substituted halide 11b and a suitable base such DIEA isadded to the resin bound amino acid and this mixture is shaken for 18-36h. The resulting mixture is washed with several portions of a suitablesolvent and is suspended and shaken in an acidic solution, such as 50%TFA/CH₂Cl₂, over several hours to cleave the acid from the resin to givea mixture of the Nα,Nα-bis-cinnamyl amino acid 11c and the Nα-cinnamylamino acid 11d.

By varying the resin bound amino acid 11a, one may obtain many of thecompounds of the invention. The following resin bound amino acids may beused in Scheme 11: alanine,N-g-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)arginine,β-(4-methyltrityl)asparagine, aspartic acid (β-t-butyl ester),S-(trityl)cysteine, γ-(4-methyltrityl)glutamine, glutamic acid(β-t-butyl ester), glycine, N-imidazolyl-(trityl)histidine, isoleucine,leucine. N-ε-(2-chlorobenzyloxycarbonyl)lysine,N-ε-(t-butoxycarbonyl)lysine, methionine, phenylalanine, proline,O-(t-butyl)serine, O-(t-butyl)threonine,N-indolyl-(t-butoxycarbonyl)tryptophan, O-(t-butyl)tyrosine, valine,β-alanine, α-aminoadipic acid, 4-aminobutanoic acid, 6-aminohexanoicacid, α-aminosuberic acid, 5-aminopentanoic acid, p-aminophenylalanine,α-aminopimelic acid γ-carboxyglutamic acid, p-carboxyphenylalanine,carnitine, citrulline, α,β-diaminopropionic acid, α,γ-diaminobutyricacid, homocitrulline, homoserine, and statine.

Scheme 12, Step A

An equivalent of an N-Fmoc-protected amino acid which is bound to aresin 11a is suspended in a suitable solvent such as DMF. This solventis removed and the nitrogen protecting group (Fmoc) is removed bystirring the resin bound amino acid with an organic base, such aspiperidine, and an addition portion of the solvent. After filtration andwashing with solvent, the resin is suspended in an appropriate solventsuch as trimethyl orthoformate (TMOF), an appropriately substitutedaldehyde 12a (5 equivalents) is added, and the mixture is shaken underN₂ overnight. This mixture is treated with a suspension of NaBH(OAc)₃ (5equivalents) in CH₂Cl₂ and shaken under N₂ overnight. After filtrationand washing with a suitable solvent, the resulting product, resin boundNα-monosubstituted amino acid 12b, is suspended and shaken in an acidicsolution, such as 50% TFA/CH₂Cl₂, over several hours to cleave the acidfrom the resin to give the Nα-cinnamyl amino acid 11d.

Scheme 12, Step B

The resin 12b is suspended in an appropriate solvent such as DMF and isfiltered. The appropriately substituted halide 12c and an appropriatebase such as DIEA are added with some additional solvent and the mixtureis shaken under N₂ for 18-36 h. The resin bound Nα,Nα-cinnamyl aminoacid 12d is isolated from the suspension and the resin is cleaved withan acidic solution as described above to give the free acid 12e.

General Procedure for the Solution-Phase Synthesis of Nα,Nα-Bis-CinnamylAmino Acids and Nα-Cinnamyl Amino Acids Scheme 13

A solution of of amino acid ester 13a, an appropriately substitutedhalide 11b, and an appropriate base such as DIEA, Na₂CO₃, or Cs₂CO₃ in asuitable solvent, such as DMF, is heated at 50-100° C. under N₂overnight, or until the starting material is exhausted, to give amixture of the Nα,Nα-bis-cinnamyl amino acid ester 13b and Nα-cinnamylamino acid ester 13c. If the side chain of R¹ contains an acid-cleavableprotecting group such as t-butylcarbamate, t-butyl ester, or t-butylether, those groups may be cleaved by, treatment with an acidic solutionsuch as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. For examples of 13b and13c where the ester group R⁴ is a primary alkyl group such as methyl orethyl, esters 13b and 13c may be independently converted to thecorresponding acids 11c and 11d by hydrolysis with an appropriate basesuch as aqueous NaOH, KOH, or LiOH. For examples of 13b and 13c wherethe ester group R⁴ is an acid-cleavable group such as t-butyl, esters13b and 13c may be independently converted to the corresponding acids11c and 11d by treatment with an acidic solution such as 30-80%TFA/CH₂Cl₂ or 2-4N HCl in EtOAc.

Scheme 14, Step A

A solution of 1 mmol of amino acid ester and 1-1.5 mmol of theappropriately substituted aldehyde 12a in 3-5 mL of TMOF was stirred atroom temperature under N₂ overnight. The solution was concentrated andused directly for the next reaction; optionally, the solution waspartitioned between EtOAc and water, washed with brine, dried overNa₂SO₄, and concentrated to give crude product, which was purified byMPLC to give mono-substituted product 14a. For examples of 14a whereinthe side chain R¹ contained an acid-cleavable protecting group such ast-butylcarbamate, t-butyl ester, or t-butyl ether, 8c was treated withan acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. Thereaction mixture was concentrated and optionally dissolved in HOAc andfreeze-dried to give the deprotected form of 14a. For examples of 14awhere the ester group R⁴ is a primary alkyl group such as methyl orethyl, esters 14a may be converted to the corresponding acids 11d byhydrolysis with an appropriate base such as aqueous NaOH, KOH, or LiOH.For examples of 14a where the ester group R⁴ is an acid-cleavable groupsuch as t-butyl, esters 14a may be converted to the corresponding acids11d by treatment with an acidic solution such as 30-80% TFA/CH₂Cl₂ or2-4N HCl in EtOAc.

Scheme 14, Step B

Amino ester 14a was dissolved in DMF, combined with 1.1-1.5 mmol of theappropriately substituted chloride or bromide 12c, and heated at 50-100°C. overnight. The reaction mixture was cooled and partitioned betweenwater and EtOAc. The organic layer was washed with water and brine,dried over Na₂SO₄, and concentrated. The crude product was purified byMPLC to give pure 14b. For examples of 14b wherein the side chain R¹contained an acid-cleavable protecting group such as t-butylcarbamate,t-butyl ester, or t-butyl ether, 8c was treated with an acidic solutionsuch as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc. The reaction mixture wasconcentrated and optionally dissolved in HOAc and freeze-dried to givethe deprotected form of 14b. For examples of 14b where the ester groupR⁴ is a primary alkyl group such as methyl or ethyl, esters 14b may beconverted to the corresponding acids 12e by hydrolysis with anappropriate base such as aqueous NaOH, KOH, or LiOH. For examples of 14bwhere the ester group R⁴ is an acid-cleavable group such as t-butyl,esters 14b may be converted to the corresponding acids 12e by treatmentwith an acidic solution such as 30-80% TFA/CH₂Cl₂ or 2-4N HCl in EtOAc.

Although the claimed compounds are useful as competitive binders to theEPO receptor, some compounds are more active than others and are eitherpreferred or particularly preferred.

The particularly preferred “R¹”s are the side chain of lysine,ornithine, arginine, aspartic acid, glutamic acid, glutamine, cysteine,methionine, serine, and threonine.

The particularly preferred “R² and R³” s are phenoxy, substitutedphenoxy, benzyloxy, and substituted benzyloxy.

The particularly preferred “R⁴ and R⁵” s are phenoxy, substitutedphenoxy, benzyloxy, and substituted benzyloxy.

The particularly preferred “W” is —CH═CH—

The particularly preferred “Q” is —CH═CH—

The particularly preferred “X” are C₁₋₅alkenyl and CH₂.

The particularly preferred “Y” are C₁₋₅alkenyl and CH₂.

The particularly preferred “n” are 1 and 2.

The particularly preferred “Z” are hydroxy, methoxy, phenethylamino,substituted phenethylamino, and —NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH—.

Pharmaceutically useful compositions the compounds of the presentinvention, may be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the compound of the presentinvention.

Therapeutic or diagnostic compositions of the invention are administeredto an individual in amounts sufficient to treat or diagnose disorders inwhich modulation of EPO receptor-related activity is indicated. Theeffective amount may vary according to a variety of factors such as theindividual's condition, weight, sex and age. Other factors include themode of administration. The pharmaceutical compositions may be providedto the individual by a variety of routes such as subcutaneous, topical,transdermal, oral and parenteral.

The term “chemical derivative” describes a molecule that containsadditional chemical moieties which are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds disclosed herein may be used alone at appropriate dosagesdefined by routine testing in order to obtain optimal inhibition of theEPO receptor or its activity while minimizing any potential toxicity. Inaddition, co-administration or sequential administration of other agentsmay be desirable.

The present invention also has the objective of providing suitabletopical, transdermal, oral, systemic and parenteral pharmaceuticalformulations for use in the novel methods of treatment of the presentinvention. The compositions containing compounds according to thisinvention as the active ingredient for use in the modulation of EPOreceptors can be administered in a wide variety of therapeutic dosageforms in conventional vehicles for administration. For example, thecompounds or modulators can be administered in such oral dosage forms astablets, capsules (each including timed release and sustained releaseformulations), pills, powders, granules, elixirs, tinctures, solutions,suspensions, syrups and emulsions, or by transdermal delivery orinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, transdermal, or intramuscular form, all using formswell known to those of ordinary skill in the pharmaceutical arts. Thecompounds of the present invention may be delivered by a wide variety ofmechanisms, including but not limited to, transdermal delivery, orinjection by needle or needle-less injection means. An effective butnon-toxic amount of the compound desired can be employed as an EPOreceptor modulating agent.

The daily dosage of the products may be varied over a wide range from0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the EPO receptor modulators are adjusted whencombined to achieve desired effects. On the other hand, dosages of thesevarious agents may be independently optimized and combined to achieve asynergistic result wherein the pathology is reduced more than it wouldbe if either agent were used alone.

Advantageously, compounds or modulators of the present invention may beadministered in a single daily dose, or the total daily dosage may beadministered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

For combination treatment with more than one active agent, where theactive agents are in separate dosage formulations, the active agents canbe administered concurrently, or they each can be administered atseparately staggered times.

The dosage regimen utilizing the compounds or modulators of the presentinvention is selected in accordance with a variety of factors includingtype, species, age, weight, sex and medical condition of the patient;the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

In the methods of the present invention, the compounds or modulatorsherein described in detail can form the active ingredient, and aretypically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents which may be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations which generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form,e.g., alcoholic solutions, topical cleansers, cleansing creams, skingels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The compounds or modulators of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels, and other suitable polymers known to those skilled in theart.

For oral administration, the compounds or modulators may be administeredin capsule, tablet, or bolus form or alternatively they can be mixed inthe animals feed. The capsules, tablets, and boluses are comprised ofthe active ingredient in combination with an appropriate carrier vehiclesuch as starch, talc, magnesium stearate, or di-calcium phosphate. Theseunit dosage forms are prepared by intimately mixing the activeingredient with suitable finely-powdered inert ingredients includingdiluents, fillers, disintegrating agents, and/or binders such that auniform mixture is obtained. An inert ingredient is one that will notreact with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intraruminal, intratracheal, or subcutaneous, either byneedle or needle-less means. The injectable formulation consists of theactive ingredient mixed with an appropriate inert liquid carrier.Acceptable liquid carriers include the vegetable oils such as peanutoil, cotton seed oil, sesame oil and the like as well as organicsolvents such as solketal, glycerol formal and the like. As analternative, aqueous parenteral formulations may also be used. Thevegetable oils are the preferred liquid carriers. The formulations areprepared by dissolving or suspending the active ingredient in the liquidcarrier such that the final formulation contains from 0.005 to 10% byweight of the active ingredient.

Topical application of the compounds or modulators is possible throughthe use of a liquid drench or a shampoo containing the instant compoundsor modulators as an aqueous solution or suspension. These formulationsgenerally contain a suspending agent such as bentonite and normally willalso contain an antifoaming agent. Formulations containing from 0.005 to10% by weight of the active ingredient are acceptable. Preferredformulations are those containing from 0.01 to 5% by weight of theinstant compounds or modulators.

The compounds of Formula I may be used in pharmaceutical compositions totreat patients (humans and other mammals) with disorders or conditionsassociated with the production of erythropoietin or modulated by the EPOreceptor. The compounds can be administered in the manner of thecommercially available product or by any oral or parenteral route(including but not limited to, intravenous, intraperitoneal,intramuscular, subcutaneous, dermal patch), where the preferred route isby injection. When the method of administration is intravenous infusion,compound of Formula I may be administered in a dose range of about 0.01to 1 mg/kg/min. For oral administration, the dose range is about 0.1 to100 mg/kg.

The pharmaceutical compositions can be prepared using conventionalpharmaceutical excipients and compounding techniques. Oral dosage formsmay be used and are elixirs, syrups, capsules, tablets and the like.Where the typical solid carrier is an inert substance such as lactose,starch, glucose, methyl cellulose, magnesium stearate, dicalciumphosphate, mannitol and the like; and typical liquid oral excipientsinclude ethanol, glycerol, water and the like. All excipients may bemixed as needed with disintegrants, diluents, granulating agents,lubricants, binders and the like using conventional techniques known tothose skilled in the art of preparing dosage forms. Parenteral dosageforms may be prepared using water or another sterile carrier.

Typically the compounds of Formula I are isolated as the free base,however when possible pharmaceutically acceptable salts can be prepared.Examples of such salts include hydrobromic, hydroiodic, hydrochloric,perchloric, sulfuric, maleic, fumaric, malic, tartaric, citric, benzoic,mandelic, methanesulfonic, hydroethanesulfonic, benzenesulfonic, oxalic,pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamicand saccharic.

In order to illustrate the invention the following examples areincluded. These examples do not limit the invention. They are only meantto suggest a method of practicing the invention. Those knowledgeable inchemical synthesis and the treatment of EPO related disorders may findother methods of practicing the invention. However those methods aredeemed to be within the scope of this invention.

BIOLOGICAL EXAMPLES

The compounds of the invention were evaluated for the ability to competewith EPO in the following immobilized EPO receptor preparation (EBP-Ig,EPO binding protein-Ig).

EBP-Ig fusion protein (as disclosed in WO97/27219 which is hereinincorporated by reference) was purified by affinity chromatography fromthe conditioned media of NSO cells engineered to express a recombinantgene construct which functionally joined the N-terminal 225 amino acidsof the human EPO receptor and an Ig heavy chain as described herein. Theinteraction of biotin and streptavidin is frequently employed to captureand effectively immobilize reagents useful in assay protocols and hasbeen employed here as a simple method to capture and immobilize EBP-Ig.EBP-Ig is initially randomly modified with an amine reactive derivativeof biotin to produce biotinylated-EBP-Ig. Use of streptavidin coatedplates allows the capture of the biotinylated EBP-Ig on the surface of ascintillant impregnated coated well (Flash plates, NEN-DuPont). Uponbinding of [¹²⁵I]EPO to the ligand binding domain, specific distancerequirements are satisfied and the scintillant is induced to emit lightin response to the energy emitted by the radioligand. Unboundradioligand does not produce a measurable signal because the energy fromthe radioactive decay is too distant from the scintillant. The amount oflight produced was quantified to estimate the amount of ligand binding.The specific assay format was suitable for the multi-well plate capacityof a Packard TopCount Microplate Scintillation counter. Compounds whichwere capable of reducing the amount of detected signal throughcompetitive binding with the radioligand were identified.

Biotinylated EBP-Ig was prepared as follows. EBP-Ig (3 mL, OD₂₈₀ 2.9)was exchanged into 50 mM sodium bicarbonate, pH 8.5 using a Centriprep10 ultrafiltration device. The final volume of the exchanged protein was1.2 mL (OD₂₈₀ 2.6, representing about 2 mg total protein). 10 μL of a 4mg/ml solution of NHS-LC-Biotin (Pierce) was added and the reactionmixture placed on ice in the dark for two hours. Unreacted biotin wasremoved by exchange of the reaction buffer into PBS in a Centriprep 10device and the protein reagent aliquoted and stored at −70° C.

Each individual binding well (200 μL) contained final concentrations of1 μg/mL of biotinylated EBP-Ig, 0.5 nM of [¹²⁵I]EPO (NEN ResearchProducts, Boston, 100 μCi/μg) and 0-500 μM of test compound (from a10-50 mM stock in 100% DMSO). All wells were adjusted to a final DMSOconcentration of 5%. All assay points were performed in triplicate andwith each experiment a standard curve for unlabelled EPO was performedat final concentration of 2000, 62, 15, 8, 4, and 0 nM. After alladditions were made, the plate was covered with an adhesive top seal andplaced in the dark at room temperature overnight. The next day allliquid was aspirated from the wells to limit analyte dependent quench ofthe signal, and the plates were counted on a Packard TOPCOUNT MicroplateScintillation Counter. Non-specific binding (NSB) was calculated as themean CPM of the 2000 nM EPO wells and total binding (TB) as the mean ofthe wells with no added unlabelled EPO. Corrected total binding (CTB)was calculated as: TB−NSB=CTB. The concentration of test compound whichreduced CTB to 50% was reported as the IC₅₀. Typically the IC₅₀ valuefor unlabelled EPO was ca. 2-7 nM and EMP1 was 0.1 μM. Table 1 lists theaverage % inhibition, and if determined the IC₅₀ and IC₃₀ values forcompounds of Formula I, where the compound numbers refer to thecompounds in the tables accompanying the preparative examples.

TABLE 1 Inhibition of EPO binding to EBP-Ig cpd % inh @ 50 μM IC₃₀ μM*IC₅₀, μM* 11 70 nd nd 12 59 nd nd 14 30 nd nd 15 48 nd nd 77 52 30 40 8232 nd nd 86 37 nd nd 100 34 nd nd 101 32 nd nd 104 78 10 30 105 70 25 35107 78 30 42 108 81 23 36 110 54 6 10 112 59 2 10 114 37 10 nd 115 35 ndnd 116 32 nd nd 117 34 nd nd 118 36 2 10 119 34 nd nd 120 35 nd nd 12145 6 nd 137 60 5 30 139 46 2 10 178 36 nd nd 179 30 nd nd 183 36 nd nd184 53 10 nd 203 37 50 nd 211 62 20 65 220 45 30 50 221 48 10 80 222 565 nd 224 51 25 50 227 48 20 50 230 42 nd nd 231 36 nd nd 235 49 20 50237 55 30 70 238 39 nd nd 239 46 8 50 243 75 2 18 244 66 1 28 246 79 1075 247 47 7 18 248 56 7 20 249 72 7 10 250 78 7 20 251 49 10 45 261 511.5 2 262 93 1 1.5 263 88 1 1.5 264 89 1.5 8 265 65 1 6 266 82 1 4 26783 2 6 268 40 nd nd 269 55 8 85 270 56 7 100 271 77 2 7 272 78 5 10 28541 nd nd 286 46 35 65 287 36 nd nd 300 57 35 145 305 48 35 225 312 45 1085 321 42 45 nd 363 33 35 220 366 38 65 nd 368 40 90 nd *nd = notdetermined

PREPARATIVE EXAMPLES

Unless otherwise noted, materials used in the examples were obtainedfrom commercial supplies and were used without further purification.Melting points were determined on a Thomas Hoover apparatus and areuncorrected. Proton nuclear magnetic resonance (¹H NMR) spectra weremeasured in the indicated solvent with tetramethylsilane (TMS) as theinternal standard using a Bruker AC-300 NMR spectrometer. NMR chemicalshifts are expressed in parts per million (ppm) downfield from internalTMS using the d scale. ¹H NMR data are tabulated in order; multiplicity,(s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), numberof protons, coupling constant in Hertz). Electrospray (ES) mass spectra(MS) were determined on a Hewlett Packard Series 1090 LCMS Engine.Elemental analyses were performed by Quantitative Technologies, Inc.(QTI), PO Box 470, Salem Industrial Park, Bldg #5, Whitehouse, N.J.08888-0470. Analytical thin layer chromatography (TLC) was done withMerck Silica Gel 60 F₂₅₄ plates (250 micron). Medium pressure liquidchromatography (MPLC) was done with Merck Silica Gel 60 (230-400 mesh).

Example 1 N,N-bis(3-Phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 96) andN-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe (cpd 334)

A solution of 500 mg (1.97 mmol) of H-Glu(O-t-Bu)OMe.HCl, 997 mg (3.45mmol) of 3-phenoxycinnamyl bromide (Jackson, W. P.; Islip, P. J.; Kneen,G.; Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and 0.89 mL(5.1 mmol, 660 mg) of DIEA in 5 mL of DMF was stirred under N₂ at roomtemperature for 40 h. The mixture was partitioned between EtOAc andwater and the organic layer was washed with water and brine. Afterdrying over Na₂SO₄, the organic solution was concentrated to give 1.24 gof orange oil. The crude residue was purified by MPLC using a solventgradient ranging from 10-30% EtOAc/hexanes to give two products. Theless polar product (cpd 96, 235 mg, 19% based on starting amino acid),was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.39 (s, 9H),2.0 (m, 2H), 2.33 (dt, 2H, J=2, 7 Hz), 3.24 (dd, 2H, J=8, 15 Hz), 3.5,(m, 3H), 3.69 (s, 3H), 6.13 (m, 2H), 6.47 (d, 2H, J=16 Hz), 6.86 (dd,2H, J=1.5, 8 Hz), 7.0-7.4 (complex, 16H); MS (ES+) m/z 634 (MH+).

The more polar product (cpd 334, 422 mg, 50% based on starting aminoacid) was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.42(s, 9H), 1.9 (m, 2H), 2.35 (t, 2H, J=7.5 Hz), 3.2-3.4 (complex, 3H),3.71 (s, 3H), 6.17 (dt, 1H, J=16, 6 Hz), 6.46 (d, 1H, J=16 Hz), 6.87(dd, 1H, J=1.5, 8 Hz), 7.01 (m, 3H), 7.10 (t, 2H, J=7.5 Hz), 7.2-7.4(complex, 3H); MS (ES+) m/z 426 (MH+). Anal. Calcd for C₂₅H₃₁NO₅: C,70.57; H, 7.34; N, 3.29. Found: C, 70.29; H, 7.14; N, 3.08.

Example 2 N-(3-Phenoxycinnamyl)Glu-OMe (cpd 325)

A solution of 95 mg (0.22 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe(cpd 334) in 3 mL of 50% TFA/CH₂Cl₂ was stirred for 2 h at roomtemperature. The mixture was concentrated and the residue was dissolvein acetic acid and freeze-dried to give 117 mg ofN-(3-phenoxycinnamyl)Glu-OMe (cpd 325) as an off-white solid; ¹H NMR(CD₃OD, 300 MHz) 2.3-2.7 (complex, 4H), 3.78 (s, 3H), 3.81 (d, 2H, J=7Hz), 4.09 (t, 1H, J=5 Hz), 6.17 (dt, 1H, J=16, 7 Hz), 6.55 (d, 1H, J=16Hz), 6.9 (m, 4H), 7.11 (t, 2H, J=7.5 Hz), 7.3 (m, 4H); MS (ES+) m/z 370(MH+), 209. Anal. Calcd for C₂₁H₂₃NO₅.C₂HF₃O₂: C, 57.14; H, 5.00; N,2.90. Found: C, 57.07; H, 5.02; N, 2.73.

Example 3 N,N-bis(3-Phenoxycinnamyl)Asp(O-t-Bu)-O-t-Bu (cpd 106)

A solution of 1.00 g (3.55 mmol) of Asp(O-t-Bu)-O-t-Bu.HCl, 2.05 g (7.1mmol) of 3-phenoxycinnamyl bromide, and 1.86 mL (10.7 mmol, 1.38 g) ofDIEA in 15 mL of DMF was heated under N₂ at 60° C. overnight. Additional3-phenoxycinnamyl bromide (1.0 g, 3.4 mmol) and DIEA (0.95 mL, 0.705 g,5.4 mmol) were added and heating was continued for an additional 14 h.The mixture was cooled and partitioned between EtOAc and water. Theorganic layer was washed twice with water, once with brine, and wasdried over Na₂SO₄. The solution was concentrated to give 3.5 g of anamber oil which was purified by MPLC using a solvent gradient rangingfrom 2.5-3% EtOAc/hexanes to afford 1.21 g of cpd 106 as a pale yellowoil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 9H), 1.48 (s, 9H), 2.49 (dd, 1H,J=7.5, 15.5 Hz), 2.70 (dd, 1H, J=7.5, 15.5 Hz), 3.26 (dd, 2H, J=7.5,14.5 Hz), 3.47 (dd, 2H, J=4, 14.5 Hz), 3.88 (t, 1H, J=7.5), 6.13 (m,2H), 6.48 (d, 2H, J=16 Hz), 6.86 (dd, 2H, J=2, 8 Hz), 7.0 (m, 6H), 7.1(m, 4H), 7.2-7.4 (complex, 6H); MS (ES+) m/z 662 (MH+).

Example 4 N,N-bis(3-Phenoxycinnamyl)Asp-OH (cpd 107)

A solution of 1.14 g (1.62 mmol) of cpd 106 in 16 mL of 50% TFA/CH₂Cl₂was stirred at room temperature for 24 h. The solution was concentratedand pumped to give 1.37 g (˜100%) cpd 107 as an amber oil; ¹H NMR(CD₃OD, 300 MHz) 3.1 (m, 2H), 4.0 (dd, 2H, J=8, 14 Hz), 4.27 (dd, 2H,J=8, 16 Hz), 4.70 (t, 1H, J=4 Hz), 6.38 (2H, dt, J=16, 8 Hz), 6.7-7.4(complex, 20H); MS (ES−) m/z 562 ([M-H]+).

Example 5 N,N-bis(4-Benzyloxybenzyl)Lys(Boc)-OMe (cpd 111) andN-(4-Benzyloxybenzyl)Lys(Boc)-OMe

A solution of 594 mg (2.0 mmol) of Lys(Boc)-OMe.HCl, 524 mg (2.25 mmol)of 4-benzyloxybenzyl chloride, 75 mg (0.5 mmol), of NaI, and 0.61 mL(3.5 mol, 452 mg) of DIEA was warmed at 50-70° C. under N₂ overnight.The mixture was cooled and partioned between EtOAc and water. Theorganic layer was washed twice with water, once with brine, and wasdried over Na₂SO₄. The organic solution was concentrated to give 0.83 gof amber oil which was purified by MPLC using a solvent gradient rangingfrom 15-40% EtOAc/hexanes to give two products. The less polar product(296 mg), cpd 111, was isolated as a pale yellow oil; ¹H NMR (CDCl₃, 300MHz) 1.28 (m, 4H), 1.43 (s, 9H), 1.70 (m, 2H), 3.03 (m, 2H), 3.28 (t,1H, J=7 Hz), 3.40 (d, 2H, J=13.5 Hz), 3.74 (s, 3H), 3.81 (d, 2H, J=13.5Hz), 5.05 (2, 4H), 6.92 (d, 4H, J=8.5), 7.23 (d, 4H, J=8.5), 7.25-7.5(complex, 10H); MS (ES+) m/z 653 (MH+).

The more polar product (406 mg), N-(4-benzyloxybenzyl)Lys(Boc)-OMe, wasisolated as a white solid; ¹H NMR (CDCl₃, 300 MHz) 1.4 (, 4H), 1.43 (s,9H), 1.65 (m, 3H), 3.08 (m, 2H), 3.23 (t, 1H, J=6.5 Hz), 3.54 (d, 1H,J=12.5 Hz), 3.71 (s, 3H), 3.73 (d, 1H, J=12.5 Hz), 5.05 (s, 2H), 6.92(d, 2H, J=8.5 Hz), 7.23 (d, 2H, J=8.5 Hz), 7.25-7.5 (complex, 5H); MS(ES+) m/z 457 (MH+).

Example 6 N-(4-Benzyloxybenzyl)-N-(3-nitrobenzyl)Lys(Boc)-OMe (cpd 113)

A solution of 374 mg (0.82 mmol) of N-(4-Benzyloxybenzyl)Lys(Boc)-OMe,221 mg (1.03 mmol) of 4-nitrobenzyl bromide, and 197 L (1.13 mmol, 146mg) of DIEA was warmed at 50-70° C. for 4 h, then at 40-50° C.overnight. After the addition of 0.2 mL of 1N aqueous HCl, the mixturewas partioned between EtOAc and water. The organic layer was washedtwice with water, once with brine, and was dried over Na₂SO₄. Theorganic solution was concentrated to give 610 mg of an amber oil whichwas purified by MPLC 1:3 EtOAc/hexanes to afford 436 mg (90%) cpd 113 asa pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.35 (m, 4H), 1.42 (s, 9H),1.75 (broad q, 2H, J=8 Hz), 3.06 (broad q, 2H, J=6 Hz), 3.28 (t, 1H,J=7.5 Hz), 3.48 (d, 1H, J=13.5 Hz), 3.66 (d, 1H, J=14.5 Hz), 3.76 (s,3H), 3.79 (d, 1H, J=13.5 Hz), 3.97 (d, 1H, J=14.5 Hz), 4.47 (broad s,1H), 5.05 (s, 2H), 6.93 (d, 2H, J=8.5 Hz), 7.22 (d, 2H, J=8.5 Hz),7.3-7.5 (complex, 6H), 7.65 (d, 1H, J=7.5 Hz), 8.09 (d, 1H, J=8 Hz),8.22 (s, 1H); MS (ES+) m/z 592 (MH+).

Example 7 N-(3-Aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe

A solution of 361 mg (0.61 mmol) of cpd 113 and 835 mg (3.7 mmol) ofSnCl₂ dihydrate was stirred under N₂ at room temperature for 6 h. Theslightly cloudy mixture was poured into 200 mL of 5% aqueous Na₂CO₃ withrapid stirring. The resulting milky suspension was extracted with three75 mL portions of CH₂Cl₂ and the combined organic layers were washedwith brine and dried over Na₂SO₄. The extracts were concentrated to give344 mg of colorless oil which was purified by MPLC using 1:2EtOAc/hexanes to provide 291 mg ofN-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe as a yellow oil; ¹HNMR (CDCl₃, 300 MHz) 1.25 (m, 4H), 1.44 (s, 9H), 1.70 (m, 2H), 3.31 (dd,1H, J=6, 9 Hz), 3.38 (d, 1H, J=14 Hz), 3.40 (d, 1H, J=13.5 Hz), 3.74 (s,3H), 3.81 (d, 1H, J=14 Hz), 3.83 (d, 1H, J=13.5 Hz), 4.52 (broad s, 1H),5.05 (s, 2H), 6.50 (broad d, 1H, J=8 Hz), 6.70 (m, 2H), 6.92 (d, 2H,J=8.5 Hz), 7.08 (t, 1H, J=7.5 Hz), 7.2-7.5 (complex, 7H); MS (ES+) m/z562 (base, MH+), 506.

Example 8N-(4-Benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys-OMe (cpd117)

A solution of 42 mg (0.075 mmol) ofN-(3-aminobenzyl)-N-(4-benzyloxybenzyl)Lys(Boc)-OMe and 12 μL (12 mg,0.15 mmol) of pyridine in 0.5 mL of 1,2-dichloroethane was combined with8.1 μL (11 mg, 0.083 mmol) and stirred under N₂ overnight. EtOAc (3 mL)was added and the solution was washed twice with 2 mL of water and 2 mLof saturated aqueous NaHCO₃. The EtOAc solution was filtered through apad of Na₂SO₄ and concentrated to give 44 mg ofN-(4-benzyloxybenzyl)-N-(3-((2-furancarbonyl)amino)benzyl)Lys(Boc)-OMe;MS (ES+) m/z 356 (MH+). The Boc-protected intermediate was stirred in 2mL of 50% TFA/CH₂Cl₂ for 2 h and was concentrated and pumped at highvacuum to provide 66 mg of cpd 117 as the bis-TFA salt; ¹H NMR (CD₃OD,300 MHz) 1.55 (m, 2H), 1.65 (m, 2H), 2.10 (m, 2H), 2.93 (t, 2H, J=7 Hz),3.68 (t, 1H, J=7 Hz), 3.78 (s, 3H), 4.20 (m, 4H), 5.09 (s, 2H), 6.66(dd, 1H, J=1.5, 3.5 Hz), 7.03 (d, 2H, J=8.5 Hz), 7.1-7.6 (complex, 11H),7.76 (m, H) 8.07 (m, 1H); MS (ES+) m/z 556 (base, MH+), 360, 197.

Example 9 N,N-bis(3-Nitrobenzyl)Asp(O-t-Bu)-O-t-Bu (cpd 62)

A solution of 0.50 mg (1.77 mmol) of Asp(O-t-Bu)-Ot-Bu.HCl, 1.17 g (5.42mmol) of 3-nitrobenzyl bromide, and 1.25 mL (0.93 g, 7.2 mmol) of DIEAin 6 mL of DMF was stirred at room temperature under N₂ for 24 h and washeated at 70-80° C. overnight. The reaction mixture was partitionedbetween EtOAc and water and the organic layer was washed twice withwater and once with brine. After drying over Na₂SO₄, the organicsolution was concentrated to give 0.86 g of a yellow oil which waspurified by MPLC using 1:9 EtOAc/hexanes to afford 0.849 g (93%) cpd 62as a pale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.43 (s, 9H), 1.57 (s,9H), 2.59 (dd, 1H, J=7.5, 16 Hz), 2.76 (dd, 1H, J=7, 16 Hz), 3.72 (t,1H, J=7.5 Hz), 3.78 (d, 2H, J=14 Hz), 3.92 (d, 2H, J=14 Hz), 7.47 (t,2H, J=8 Hz), 7.67 (d, 2H, J=7.5 Hz), 8.09 (broad d, 2H J=8 Hz), 8.16(broad s, 2H); MS (ES+) m/z 538 (MNa+), 516 (base, MH+), 460, 404, 237.

Example 10 N,N-bis(3-Aminobenzyl)Asp(O-t-Bu)-O-t-Bu

A solution of 0.644 g (1.25 mmol) of cpd 62 and 2.82 g (12.5 mmol) ofSnCl₂.2H₂O in 12 mL of absolute EtOH was refluxed for 1.5 h. The mixturewas cooled and poured into 300 mL of 5% aqueous Na₂CO₃ with rapidstirring. The cloudy mixture was extracted with three 150 mL portions ofCH₂Cl₂ and the organic extracts were washed with brine and dried overNa₂SO₄. The CH₂Cl₂ solution was concentrated to afford 210 mg (37%) ofN,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a cloudy yellow oil whichwas used without purification; ¹H NMR (CDCl₃, 300 MHz) 1.40 (s, 9H),1.52 (s, 9H), 2.48 (dd, 1H, J=7, 16 Hz), 2.76 (dd, 1H, J=8, 16 Hz), 3.48(d, 2H, J=14 Hz), 3.55 (m, 1H), 3.73 (d, 2H, J=14 Hz), 6.56 (broad d, 2HJ=8 Hz), 6.70 (broad s, 2H), 6.77 (d, 2H, J=7.5 Hz), 7.08 (t, 2H, J=8Hz); MS (ES+) m/z 478 (MNa+), 456 (base, MH+), 400, 344.

Example 11 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu

To a solution of 109 mg (0.24 mmol) ofN,N-bis(3-aminobenzyl)Asp(O-t-Bu)-O-t-Bu, 29 mg (0.24 mmol) of DMAP, 125μL (93 mg, 0.72 mmol) of DIEA in 1 mL of CH₂Cl₂ was added 95 μL (111 mg,0.72 mmol) of 4-methylbenzoyl chloride. The solution was stirred underN₂ overnight and was then partitioned between EtOAc and water. Theorganic layer was washed with saturated aqueous NaHCO₃ and brine, driedover Na₂SO₄, and concentrated to give 177 mg of yellow oil. The crudematerial was purified by MPLC using a solvent gradient ranging from20-25% EtOAc/hexanes to provide 87 mg ofN,N-bis(3-(4-methylbenzoyl)aminobenzyl)Asp(O-t-Bu)-O-t-Bu as a paleyellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.36 (s, 9H), 1.55 (s, 9H), 2.35 (s,6H), 2.53 (dd, 1H, J=6, 16 Hz), 2.76 (dd, 1H, J=9, 16 Hz), 3.69 (d, 2H,J=14), 3.77 (dd, 1H, J=6, 9 Hz), 3.83 (d, 2H, J=14), 7.01 (m, 6H), 7.26(t, 2H, J=8 Hz), 7.59 (m, 6H), 8.11 (s, 2H), 8.49 (s, 2H); MS (ES+) m/z714 (MNa+), 692 (base, MH+), 636, 580.

Example 12 N,N-bis(3-(4-Methylbenzoyl)aminobenzyl)Asp-OH (cpd 64)

A solution of 87 mg (0.13 mmol) ofN,N-bis(3-(4-methylbenzoyl)amino-benzyl)Asp(O-t-Bu)-O-t-Bu in 1 mL of50% TFA/CH₂Cl₂ was stirred overnight. The mixture was concentrated andthe residue was dissolved in HOAc and freeze-dried to afford 77 mg cpd64 as a white solid; ¹H NMR (CD₃OD, 300 MHz) 2.40 (s, 6H), 2.85 (dd, 1H,J=6, 16.5 Hz), 2.98 (dd, 1H, J=8, 16.5 Hz), 4.02 (d, 2H, J=13.5 Hz),4.08 (d, 4H, J=13.5 Hz), 4.10 (t, 1H, J=6.5 Hz), 7.22 (m, 6H), 7.34 (t,2H, J=7.5 Hz), 7.60 (broad d, 2H, J=9 Hz), 7.76 (d, 4H, J=8 Hz), 7.88(broad s, 2H); MS (ES+) m/z 580 (base, MH+).

Example 13 [N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

To a solution of 1.69 g (5.0 mmol) of N-Cbz-Glu(O-t-Bu)-OH, 0.365 mL(0.371 g, 2.5 mmol) of 1,8-diamino-3,6-dioxaoctane, 0.743 g (5.5 mmol)of HOBT, and 1.05 mL (0.776 g, 6.0 mmol) of DIEA in 15 mL of CH₂Cl₂ wasadded 1.05 g (5.5 mmol) of EDCI in one portion. After stirring at roomtemperature under N₂ overnight, the mixture was partitioned betweenEtOAc and 10% aqueous citric acid. The organic layer was washed withwater, saturated NaHCO₃, and brine, dried over Na₂SO₄, and concentratedto give 1.87 g of (N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ as a colorless oil;¹H NMR (CD₃OD, 300 MHz) 1.43 (s, 18H), 1.85 (m, 2H), 2.05 (m, 2H), 2.31(t, 4H, J=8 Hz), 3.37 (t, 4H, J=5 Hz), 3.52 (t, 4H, J=5 Hz), 3.58 (s,4H), 4.15 (m, 2H), 5.09 (dd, 4H, J=12, 16 Hz), 7.30 (m, 10H); MS (ES+)m/z 809 (base, MNa+), 787 (MH+).

Example 14 [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

Ammonium formate (0.78 g, 12.4 mmol) and 0.16 g of 10% palladium oncarbon were added to a solution of (N-Cbz-Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ in12 mL of MeOH and the resulting suspension was stirred under N₂ at roomtemperature overnight. The mixture was diluted with CH₂Cl₂ and filteredthrough a Celite pad. The solids were washed thoroughly with CH₂Cl₂ andthe combined organic filtrates were concentrated to dryness. The residuewas partitioned between CH₂Cl₂ and saturated aqueous NaHCO₃, washed withbrine, dried over Na₂SO₄, and concentrated to give 1.13 g of(Glu(O-t-Bu)-NHCH₂CH₂OCH₂)₂ as a colorless oil; 1.44 (s, 18H), 1.81 (m,2H), 2.08 (m, 2H), 2.35 (m, 4H), 3.39 (dd, 2H, J=5, 7.5 Hz), 3.47 (t,4H, J=5 Hz), 3.58 (t, 4H, J=5 Hz), 7.53 (m, 2H).

Example 15 [N,N-bis(4-Benzyloxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ (cpd245)

A solution of 199 mg (0.384 mmol) of [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂, 403 mg(1.73 mmol) of 4-benzyloxybenzyl chloride, 30 mg (0.2 mmol) of NaI, and334 L (248 mg, 1.92 mmol) of DIEA was stirred under N₂ at roomtemperature for several days. The solution was partitioned between EtOAcand water and the organic layer was washed three times with water andonce with brine. After drying over Na₂SO₄, the solution was concentratedto give 528 mg of yellow oil which was purified by MPLC using a solventgradient ranging from 42-50% EtOAc/hexanes to afford 318 mg (64%) of cpd245 as a white foam; ¹H NMR (CDCl₃, 300 MHz) 1.42 (s, 18H), 2.01 (m,4H), 2.38 (m, 2H), 2.55 (m, 2H), 3.03 (dd, 2H, J=5, 8 Hz), 3.31 (m, 2H),3.4-3.6 (complex, 18H), 4.99 (s, 8H), 6.89 (d, 8H, J=8.5), 7.1-7.4complex, 30H).

Example 16 [N,N-bis(4-Benzyloxybenzyl)GluNHCH₂CH₂OCH₂]₂ (cpd 246)

A solution of 219 mg (0.168 mmol) of cpd 245 in 2 mL of 33% TFA/CH₂Cl₂was stirred ad room temperature overnight. The mixture was concentratedto give a crude product which was dissolved in HOAc and freeze-dried toafford 251 mg of cpd 246 as an amber oil; ¹H NMR (CD₃OD, 300 MHz)2.1-2.6 (complex, 8H), 3.3-3.6 (complex, 8H), 3.57 (s, 4H), 3.78 (m,2H), 4.25 (broad d, 4H, J=14 Hz), 4.36 (broad d, 4H, J=14 Hz), 5.09 (s,8H), 7.03 (d, 8H, J=8 Hz), 7.3-7.5 (complex, 28H); MS (ES+) m/z 1192(MH+), 995, 596, 197 (base).

Example 17 [N-(3-Phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]2

A solution of 680 mg (0.76 mmol) of [Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ and 278μL (317 mg, 1.6 mmol) of 3-phenoxybenzaldehyde in 3 mL of TMOF wasstirred overnight at room temperature under N₂. The mixture wasconcentrated and pumped at high vacuum to give a colorless oil which wasdissolved in 3 mL of CH₂Cl₂ and treated with 678 mg (3.2 mmol) ofNaBH(OAc)₃. After stirring under N₂ for 2 days, 50 mL of saturatedaqueous NaHCO₃ was added and the mixture was extracted with CH₂Cl₂. Theorganic layers were combined, dried over Na₂SO₄, and concentrated andthe crude product (1.01 g) was purified by MPLC using a solvent gradientranging from 2-4% MeOH/CH₂Cl₂ to afford 490 mg of[N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ as a colorless oil; ¹HNMR (CDCl₃, 300 MHz) 1.41 (s, 18H), 1.89 (m, 4H), 2.31 (m, 4H), 3.12 (t,2H, J=6 Hz), 3.45 (m, 8H), 3.55 (s, 4H), 3.60 (d, 2H, J=13.5 Hz), 3.73(d, 2H, J=13.5 Hz), 6.86 (dd, 2H, J=1.5, 8 Hz), 7.00 (m, 8H), 7.2-7.4(complex, 8H); MS (ES+) m/z 883 (MH+), 589, 442, 414, 386 (base), 183.

Example 18[N-(3-Nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

DIEA (269 μL, 199 mg, 1.54 mmol), 3-nitrobenzyl bromide (322 mg, 1.49mmol), and [N-(3-phenoxybenzyl)Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ (482 mg, 0.546mmol) were combined in 2 mL of DMF and heated at 60-70° C. under N₂ for2 days. The reaction mixture was cooled and partitioned between 100 mLof EtOAc and water. The organic layer was washed with three times withwater and once with brine, dried over Na₂SO₄, and concentrated to give661 mg (˜100%) of[N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ whichwas used without purification; MS (ES+) m/z 1154 (MH+), 577, 130 (base).

Example 19[N-(3-Aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂

A solution of 661 mg (0.54 mmol) of crude[N-(3-nitrobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ and2.71 g (12.0 mmol) of SnCl₂. 2H₂O in 20 mL of absolute EtOH was refluxedunder N₂ for 30 min. The cooled solution was poured into 500 mL of 2.5%aqueous Na₂CO₃ with rapid stirring and the resulting cloudy mixture wasextracted thoroughly with EtOAc. The slightly cloudy organic extractswere washed twice with brine, dried over Na₂SO₄, anc concentrated togive 604 mg of yellow oil which was purified by MPLC using 3%MeOH/CH₂Cl₂ to provide 350 mg (59%) of[N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂ as apale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.41 (s, 18H), 1.97 (m, 4H),2.25 (m, 4H), 2.48 (m, 4H), 3.03 (dd, 2H, J=5, 8 Hz), 3.30 (m, 2H),3.4-3.8 (complex, 24H), 6.47 (d, 2H, J=7.5 Hz), 6.65 (m, 4H), 6.85 (d,2H, J=9.5 Hz), 6.9-7.15 (complex, 12H), 7.2-7.4 (complex, 8H); MS (ES+)m/z 1094 (MH+), 547 (base).

Example 20[N-(3-Phenoxybenzyl)-N-(3-(pentanoylamino)benzyl)-Glu-NHCH₂CH₂OCH₂]₂(cpd 247)

Pentanoyl chloride (16 uL, 16 mg, 0.136 mmol) was added dropwise to asolution of 68 mg (0.062 mmol) of[N-(3-aminobenzyl)-N-(3-phenoxybenzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂, 20 μL(20 mg, 0.25 mmol) of pyridine in 0.3 mL of 1,2-dichloroethane. Themixture was shaken under N₂ overnight and was then partitioned betweenEtOAc and water. The organic layer was washed with saturated aqueousNaHCO₃, dried over Na₂SO₄, and concentrated to give 77 mg of[N-(3-phenoxybenzyl)-N-(3-(pentanoylamino)benzyl)-Glu(O-t-Bu)-NHCH₂CH₂OCH₂]₂;MS (ES+) m/z 1073, 575 (base, MH+/2). The crude product was dissolved in1 mL of 50% TFA/CH₂Cl₂ and allows to stand overnight. The solution wasconcentrated and the resulting oil was dissolved in HOAc andfreeze-dried to provide 82 mg of cpd 247; ¹H NMR (CD₃OD, 300 MHz) 3.98(t, 6H, J=7.5 Hz), 1.39 (sextet, 4H, J=7.5 Hz), 1.66 (quintet, 4H, J=7.5Hz), 1.65 (m, 2H), 1.78 (m, 2H), 2.35 (t, 4H, J=7.5 Hz), 2.45 (m, 4H),3.38 (m, 4H), 3.50 (t, 2H, J=5), 3.57 (m, 4H), 4.10 (broad s, 8H),6.9-7.25 (complex, 14H), 7.25-7.4 (complex, 10H), 7.71 (s, 2H); MS (ES+)m/z 1150 (MH+), 575 (base).

Example 21 [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N

A solution of 1.0 g (2.63 mmol) of N-Cbz-Lys(Boc)OH, 0.131 mL (0.128 g,0.876 mmol) of tris(2-aminoethyl)amine, 0.391 g (2.98 mmol) of HOBt,0.555 g (2.89 mmol) of EDCI, and 0.55 mL (0.408 g, 3.16 mmol) of DIEA in5 mL of CH₂Cl₂ was stirred under N₂ at room temperature overnight. Themixture was diluted with EtOAc and washed with 10% aqueous citric acid,saturated aqueous NaHCO₃, and brine. The solution was dried over Na₂SO₄and concentrated to give 0.872 g of [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N as anoff-white solid; ¹H NMR (CD₃OD, 300 MHz) 135 (m, 12H), 1.40 (s, 27H),1.60 (m, 3H), 1.72 (m, 3H), 2.51 (m, 6H), 2.99 (m, 6H), 3.10 (m, 3H),3.21 (m, 3H), 4.12 (m, 3H), 5.00 (d, 3H, J=12.5 Hz), 5.08 (d, 3H, J=12.5Hz), 7.29 (m, 15H); MS (ES+) m/z 1243 (base, MH+), 567, 467.

Example 22 [Lys(Boc)-NHCH₂CH₂]₃N

A solution of 0.841 g (0.682 mmol) [N-Cbz-Lys(Boc)-NHCH₂CH₂]₃N, 0.252 gof 10% Pd—C, and 0.774 g (12.3 mmol) of ammonium formate in 10 mL ofMeOH was stirred for 5 h at room temperature under N₂. The mixture wasfiltered through a Celite pad, the solids were washed with CH₂Cl₂, andthe reslulting solution was concentrated to dryness. The residue waspartitioned between CH₂Cl₂ and brine; the organic layer was dried overNa₂SO₄ and concentrated to provide 0.191 g of [Lys(Boc)-NHCH₂CH₂]₃N asan off-white solid; ¹H NMR (CD₃OD, 300 MHz) 1.40 (s, 27H), 1.45 (m,12H), 1.75 (m, 6H), 2.62 (m, 6H), 3.01 (m, 6H), 3.28 (m, 6H), 3.64 (m,3H); MS (ES+) m/z 853 (MNa+), 831 (MH+), 266 (base).

Example 23 [N,N-bis(3-Phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N

A solution of 65 mg (0.078 mmol) of [Lys(Boc)-NHCH₂CH₂]₃N, 120 μL (140mg, 0.70 mmol) of 3-phenoxybenzaldehyde, and 71 μL (65 mg, 0.70 mmol) ofborane-pyridine complexin 3 mL of absolute EtOH was stirred for 4 daysat room temperature under N₂. The mixture was concentrated to drynessand partitioned between water and CH₂Cl₂. The organic layer wasconcentrated to give a yellow oil which was purified by MPLC using 2.5%MeOH/CH₂Cl₂ to give 78 mg of[N,N-bis(3-phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N as a yellow oil; MS (ES+)m/z 872 (base, [M—C₁₃H₁₂O)/2]+), 611, 443.

Example 24 [N,N-bis(3-Phenoxybenzyl)Lys-NHCH₂CH₂]₃N (cpd 277)

A solution of 78 mg (0.048 mmol) of[N,N-bis(3-phenoxybenzyl)Lys(Boc)-NHCH₂CH₂]₃N in 2 mL of 50% TFA/CH₂Cl₂was stirred for 2 h at room temperature. The mixture was diluted withCH₂Cl₂, washed with water and 5% Na₂CO₃, and concentrated to give 57 mgof cpd 277 as an off-white foam; ¹H NMR (CD₃OD, 300 MHz) 1.35 (m, 6H),1.52 (m, 6H), 1.76 (m, 6H), 2.75 (m, 6H), 3.19 (m, 6H), 3.40 (m, 6H),3.60 (m, 9H), 3.77 (m, 6H), 6.79 (d, 6H, J=8 Hz), 693 (m, 24H), 7.05 (m,6H), 7.19 (m, 6H), 7.29 (m, 12H); MS (ES+) m/z 813 ([MH₂/2]+), 721, 542(base, [MH/3]+).

Example 25 N,N-bis(3-Phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290) andN-(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352)

A solution of 423 mg (2.0 mmol) of H-Ser(t-Bu)OMe.HCl, 1.01 g (3.5 mmol)of 3-phenoxycinnamyl bromide (Jackson, W. P.; Islip, P. J.; Kneen, G.;Pugh, A.; Wates, P. J. J.Med.Chem. 31 1988; 499-500), and 0.87 mL (5.0mmol, 650 mg) of DIEA in 6 mL of DMF was stirred under N₂ at roomtemperature for 20 h. The mixture was partitioned between EtOAc andwater and the organic layer was washed with water and brine. Afterdrying over Na₂SO₄, the organic solution was concentrated to give 0.98 gof yellow oil. The crude residue was purified by MPLC using a solventgradient ranging from 10-30% EtOAc/hexanes to give two products. Theless polar product (168 mg, 14% based on starting amino acid),N,N-bis(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290), was isolated as apale yellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.15 (s, 9H), 3.35 (dd, 2H,J=7, 14.5 Hz), 3.53 (dd, 2H, J=5.5, 14.5 Hz), 3.6-3.8 (complex, 3H),3.69 (s, 3H), 6.18 (dt, 2H, J=16, 6.5 Hz), 6.49 (d, 2H, J=16 Hz), 6.86(dd, 2H, J=2, 8 Hz), 6.9-7.4 (complex, 16H); MS (ES+) m/z 614, 592 (MH+,base), 406, 384, 209.

The more polar product (354 mg, 46% based on starting amino acid),N-(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352), was isolated as a paleyellow oil; ¹H NMR (CDCl₃, 300 MHz) 1.15 (s, 9H), 1.98 (broad s, 1H),3.32 (ddd, 1H, J=1.2, 6.5, 14 Hz), 3.4-3.7 (complex, 4H), 3.72 (s, 3H),6.21 (dt, 1H, J=16, 6.5 Hz), 6.48 (d, 1H, J=16 Hz), 6.88 (dd, 1H, J=1.5,8 Hz), 7.0-7.4 (complex, 8H); MS (ES+) m/z 789 (2M+Na+), 384 (MH+,base), 209.

Example 26 N,N-Bis(3-phenoxycinnamyl)Ser-OMe (cpd 299)

N,N-Bis(3-phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 290, 168 mg, 0.284 mmol)was stirred in 3 mL of 50% TFA/CH₂Cl₂ under N₂ overnight. The solventwas removed using a rotary evaporator and the crude residue waspartitioned between EtOAc and saturated aqueous NaHCO₃. After washingwith brine and drying over Na₂SO₄, the organic layer was concentratedusing a rotary evaporator and the crude product (134 mg) was purified byMPLC using 30% EtOAc/hexanes to give 44 mg (29%) ofN,N-bis(3-phenoxycinnamyl)Ser-OMe (cpd 299) as a colorless oil; ¹H NMR(CDCl₃, 300 MHz) 1.6 (broad s, 2H), 3.38 (dd, 2H, J−8, 12 Hz), 3.4-3.9(complex, 5H), 3.72 (s, 3H), 6.13 (dt, 2H, J=16, 7 Hz), 6.50 (d, 2H,J=16 Hz), 6.8-7.4 (complex, 18H); MS (ES+) m/z 536 (MH+).

Example 27 N,N-Bis(3-phenoxycinnamyl)Ser-OH (cpd 300)

N,N-Bis(3-phenoxycinnamyl)Ser-OMe (cpd 299, 44 mg, 0.082 mmol) wasdissolved in 0.2 mL of MeOH and was stirred with 0.090 mL of 1N aqueousNaOH. When TLC analysis revealed that starting material had beenconsumed, the solvent was removed by rotary evaporation and the residuewas lyophilized from acetic acid to give 42 mg (88%) ofN,N-bis(3-phenoxycinnamyl)Ser-OH acetate (cpd 300) as a sticky yellowsolid; ¹H NMR (methanol-d₄, 300 M) 1.97 (s, 3H), 3.3-4.2 (complex, 7H),6.80 (d, 2H, J=16 Hz), 6.9-7.4 (complex, 18H); MS (ES+) m/z 522 (MH+),209.

Example 28 N-(3-Phenoxycinnamyl)Ser-OMe (cpd 346)

N-(3-Phenoxycinnamyl)Ser(t-Bu)-OMe (cpd 352, 268 mg, 0.699 mmol) wasstirred in 3 mL of 50% TFA/CH₂Cl₂ under N₂ overnight. The solvent wasremoved using a rotary evaporator and the crude residue (256 mg) waspurified by MPLC using EtOAc to give 137 mg (60%) ofN-(3-phenoxycinnamyl)Ser-OMe (cpd 346) as a colorless oil; ¹H NMR(CDCl₃, 300 MHz) 2.2 (broad s, 2H), 3.36 (dd, 1H, J=6, 14 Hz), 3.4-3.5(complex, 2H), 3.62 (dd, 1H, J=6.5, 11 Hz), 3.74 (s, 3H), 3.80 (dd, 1H,J=4.5, 11 Hz). 6.19 (dt, 1H, J=16, 6.5 Hz), 6.48 (d, 1H, J=6 Hz), 6.88(dd, 1H, J=1.5, 8 Hz), 7.0-7.4 (complex, 8H); MS (ES+) m/z 677 (2M+Na+),350 (M+Na+), 328 (MH+), 209 (base).

Example 29 N-(3-phenoxycinnamyl)Ser-OH (cpd 347)

N-(3-Phenoxycinnamyl)Ser-OMe (cpd 346, 110 mg, 0.336 mmol) was dissolvedin 1.5 mL of MeOH and was stirred with 0.50 mL of 1N aqueous NaOH. WhenTLC analysis revealed that starting material had been consumed, thesolvent was removed by rotary evaporation. The residue was dissolved inwater and acidified to pH 7-8 with 1N aqueous HCl; the resulting solidswere filtered, washed with water, and dried to give 71 mg of whitepowder. The insoluble powder was dissolved in TFA and, after removal ofexcess TFA by rotary evaporation, lyophilized from acetic acid to give82 mg (57%) of N-(3-phenoxycinnamyl)Ser-OH trifluoroacetate (cpd 347) asan amber oil; ¹H NMR (methanol-d₄, 300 MHz) 3.88 (d, 2H, J=7H), 4.0-4.2(complex, 3H), 6.27 (dt, 1H, J=16, 6.5), 6.83 (d, 1H, J=16 Hz), 6.9-7.4(complex, 9H); MS (ES+) m/z 314, (MH+), 209.

Example 30 N-(3-Phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd 337)

A mixture of 249 mg (0.585 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OMe(cpd 334) in 3 mL of MeOH was sonicated to speed dissolution, and theresulting solution was treated with 0.585 mL of 1N aqueous NaOH. Afterstirring overnight, the MeOH was removed using a rotary evaporator andthe residue was dissolved in water. Acidification with 0.64 mL of 1Naqueous HCl produced a 250 mg of solid material that was triturated withEt₂O to give 111 mg (46%) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OH (cpd337) as a white solid; ¹H NMR (300 MHz, methanol-d₄) 1.43 (s, 9H),1.9-2.2 (complex, 2H), 2.46 (t, 2H, J=7 Hz), 3.57 (dd, 1H, J=5, 7 Hz),3.78 (dd, 1H, J=7, 13.5 Hz), 3.82 (dd, 1H, J=7, 13.5 Hz), 6.28 (dt, 1H,J=16, 7 Hz), 6.81 (d, 1H, J=16 Hz), 6.9-7.5 (complex, 9H); MS (ES+) m/z412 (MH+, base), 356, 209. Anal. Calcd for C₂₄H₂₉NO₅.0.4 H₂O: C, 68.55;H, 7.04; N, 3.24. Found: C, 68.89; H, 7.04; N, 3.24.

Example 31 N-(3-Phenoxycinnamyl)Glu-OH (cpd 326)

A mixture of 85 mg (0.21 mmol) of N-(3-phenoxycinnamyl)Glu(O-t-Bu)-OH(cpd 337) in was stirred in 1 mL of 50% TFA/CH₂Cl₂ for 1 h. Aftersolvent removal using a rotary evaporator, the residue was dissolved inacetic acid and freeze-dried to give 75 mg (76%) ofN-(3-phenoxycinnamyl)Glu-OH tifluoroacetate (cpd 326) as a fluffy whitesolid; ¹H NMR (300 MHz, methanol-₄) 2.0-2.4 (complex, 2H), 2.55 (m, 2H),3.84 (d, 2H, J=7 Hz), 3.96 (dd, 1H, J=5, 7 Hz, 6.24 (dt, 1H, J=16, 7Hz), 6.84 (d, 1H, J=16 Hz), 6.9-7.4 (complex, 9H); MS (ES+) m/z 356(MH+), 209 (base).

TABLE 2

R¹ (amino cpd % inh acid side chain) R² R³ W, Q 11 70 Asn, Asp, Gln, Glu3-PhO CH═CH 12 59 Cys, Met, Ser, Thr 3-PhO CH═CH 13 nd Arg, Gly, His,Pro 3-PhO CH═CH 14 30 Lys(2-Cl—Cbz), 3-PhO CH═CH Phe, Trp, Tyr 15 48Ala, Ile, Leu, Val 3-PhO CH═CH 16 nd Glu, Asp 2,3-benzo CH═CH 17 nd Cys,Met 2,3-benzo CH═CH 18 nd Ser, Thr 2,3-benzo CH═CH 19 nd His, Arg(Mtr)2,3-benzo CH═CH 20 nd Pro, Gly 2,3-benzo CH═CH 21 nd Phe, Tyr 2,3-benzoCH═CH 22 nd Trp, 2,3-benzo CH═CH Lys(2-Cl—Cbz) 23 nd Ile, Ala 2,3-benzoCH═CH 24 nd Val, Leu 2,3-benzo CH═CH 25 nd Asn, Lys 2,3-benzo CH═CH 26nd Ala, Ile 3,4-benzo CH═CH 27 nd Arg(Mtr), 3,4-benzo CH═CHLys(2-Cl—Cbz) 28 nd Asp, Glu 3,4-benzo CH═CH 29 nd Cys, Met 3,4-benzoCH═CH 30 nd Gly, Pro 3,4-benzo CH═CH 31 nd His, Lys 3,4-benzo CH═CH 32nd Leu, Val 3,4-benzo CH═CH 33 nd Lys(2-Cl—Cbz), 3,4-benzo CH═CH Phe 34nd Ser, Thr 3,4-benzo CH═CH 35 nd Trp, Tyr 3,4-benzo CH═CH

TABLE 3

EPO/EBP-Ig cpd % inh @ 50 μM R¹ R² R⁹ W, Q MS MH+ 36 nd CH₃ 4-CF₃ HCH═CH 458 37 19 H 4-CF₃ H CH═CH 430 38 nd (CH₂)₄NH(2-Cl—Cbz) 4-F H CH═CH448 40 nd CH₃ 4-F H CH═CH 223 41 nd CH₂CO₂H 4-F H CH═CH 266 42 ndCH₂CH₂CO₂H 4-F H CH═CH 281 43 nd (CH₂)₃NHC(═NH)NH₂ 4-F H CH═CH 308 45 ndPhCH₂ 4-F H CH═CH 299 46 nd 4-HO—PhCH₂ 4-F H CH═CH 315 47 nd CH₂OH 4-F HCH═CH 238 48 nd CH(OH)CH₃ 4-F H CH═CH 253 49 1 (CH₂)₃NHC(═NH)NH₂ H H S419 50 −6 (CH₂)₄NH₂ H H S 391 51 nd CH(CH₃)CH₂CH₃ H H S 376 52 21CH₂CH₂CO₂H H H S 392 53 14 CH₂CO₂H H H S 378 54 18 CH₃ H H S 334 55 4CH₂CH₂CONH₂ H H S 391 56 nd (CH₂)₄NHCbz H Me S 539 57 0 (CH₂)₄NHCbz HCH₂Ph S 615 58 nd CH₂(indol-3-yl) H Me S 463 59 26 CH₂CH₂CO₂-t-Bu H Me S462 60 9 CH₂CH₂CO₂Et H Me S 434 61 14 CH₂CH₂CO₂H H Me S 406

TABLE 4

EPO/EBP-Ig cpd % inh @ 50 μM R^(a) R² R⁴ R⁹ MS, MH+ 62 nd t-Bu NO₂ NO₂t-Bu 516 63 20 H PhCH₂NH PhO H 511 64 −4 H 4-MePhCONH 4-MePhCONH H 58065 −7 H 4-MePhSO₂NH 4-MePhSO₂NH H 652 66 −16 H 3-ClPhCH₂NH PhO H 546 67−8 H 3-BrPhCH₂NH PhO H 590 68 −13 H 2-FPhCH₂NH PhO H 529 69 −13 H2-MePhCH₂NH PhO H 525 70 −8 H 4-FPhCH₂NH PhO H 529 71 −6 H 3-ClPhCH₂NH4-Me—PhO H 560 72 −14 H F₅—PhCH₂NH 4-Me—PhO H 615 73 −13 H 2-FPhCH₂NH4-Me—PhO H 543 74 −7 H 3-CNPhCH₂NH 4-Me—PhO H 550 75 −2 H PhCH₂NH4-Me—PhO H 525

TABLE 5

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁹ n MH+ 76 25 t-BuPhO H PhO H t-Bu 1 636 77 52 H PhO H PhO H H 1 524 78 nd H H 4-MePhCONHH BnO H 2 593 79 nd H H n-BuCONH H BnO H 2 559 80 nd H H 2-naphthyl CONHH BnO H 2 629 81 nd H H 2-furyl CONH H BnO H 2 569 82 32 H H4-MeO—PhCONH H BnO H 2 609 83 18 H H HO₂C(CH₂)₃CONH H BnO H 2 589 84 14H H C₂F₅CONH H BnO H 2 621 85 20 H H CF₃CONH H BnO H 2 571 86 37 H H4-pyridyl-CONH H BnO H 2 580 87 23 H H 4-MePhSO₂NH H BnO H 2 629 88 10.3H H HO₂CCH₂(1,1- H BnO H 2 643 cyclopentyl) CH₂CONH 89 22 H H PhOCONH HBnO H 2 595 90 29 H H 4-Ph—PhCONH H BnO H 2 655 91 19 H H 4-NO₂—PhCONH HBnO H 2 624

TABLE 6

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R³ R⁴ R⁵ R⁶ R⁹ MH+ 92 20 H H HH H 2 Me 394 93 20 t-Bu H H H H 2 Me 450 94 25 Et H H H H 2 Me 422 95 15t-Bu 2,3-benzo 2,3-benzo 2 Me 550 96 −5 t-Bu PhO H PhO H 2 Me 634 97 14t-Bu 3,4-benzo 3,4-benzo 2 H 536 98 12 t-Bu H Ph H Ph 2 Me 602 99 13t-Bu 3,4-di-Cl—PhO H 3,4-di-Cl—PhO H 2 Me 772 100 34 H H Ph H Ph 2 Me546 101 32 H 3,4-di-Cl—PhO H 3,4-di-Cl—PhO H 2 Me 716 102 5 t-Bu4-t-Bu—PhO H 4-t-Bu—PhO H 2 t-Bu 789 103 17 t-Bu 3-CF3—PhO H 3-CF3—PhO H2 t-Bu 812 104 78 H 4-t-Bu—PhO H 4-t-Bu—PhO H 2 H 676 105 70 H 3-CF3—PhOH 3-CF3—PhO H 2 H 700 106 20 t-Bu PhO H PhO H 1 t-Bu 662 107 78 H PhO HPhO H 2 H  562* 108 81 H PhO H PhO H 1 H 550 *[M − H]⁻

TABLE 7

EPO/ EBP-Ig % inh @ 50 MS, cpd μM R^(a) R² R³ R⁴ R⁵ R⁹ MH+ 109 7 Boc BnOH BnO H Me 653 110 54 H H BnO H BnO Me 553 111 5 Boc H BnO H BnO Me 653112 59 H BnO H BnO H Me 553 113 24 Boc H BnO NO₂ H Me 592 114 37 H H BnONO₂ H Me 492 115 35 H H BnO NH₂ H Me 462 116 32 H H BnO n-BuCONH H Me546 117 34 H H BnO 2-furylCONH H Me 556 118 36 H H BnO 4-MePhCONH H Me580 119 34 H H BnO i-Pr—CONH H Me 532 120 35 H H BnO 4-pyridyl- H Me 567CONH 121 45 H H BnO 2-naphthyl- H Me 616 CONH 122 nd Boc PhCH₂NH HPhCH₂NH H Me 651 123 nd Boc 2-MePhCH₂NH H 2-MePhCH₂NH H Me 679 124 ndBoc 4-MeO—PhCH₂NH H 4-MeO—PhCH₂NH H Me 711 125 nd Boc 3,4-di-MeO—PhCH₂NHH 3,4-di-MeO—PhCH₂NH H Me 771 126 nd Boc —NH₂ H —NH₂ H Me 417 127 nd HPhCH₂NH H PhCH₂NH H Me 551 128 nd H 2-MePhCH₂NH H 2-MePhCH₂NH H Me 579129 nd H 4-MeO—PhCH₂NH H 4-MeO—PhCH₂NH H Me 611 130 nd H3,4-di-MeO—PhCH₂NH H 3,4-di-MeO—PhCH₂NH H Me 671 131 nd H PhCH₂CH₂NH HPhCH₂CH₂NH H Me 579 132 nd HO₂CCH₂CH₂CO PhCH₂NH H PhCH₂NH H Me 651 133nd HO₂CCH₂CH₂CO 2-MePhCH₂NH H 2-MePhCH₂NH H Me 679 134 nd HO₂CCH₂CH₂CO4-MeO—PhCH₂NH H 4-MeO—PhCH₂NH H Me 711 135 nd HO₂CCH₂CH₂CO3,4-di-MeO—PhCH₂NH H 3,4-di-MeO—PhCH₂NH H Me 771 136 nd HO₂CCH₂CH₂COPhCH₂CH₂NH H PhCH₂CH₂NH H Me 679

TABLE 8

EPO/EBP-Ig MS, cpd % inh @ 50 μM R^(a) R² R⁴ R⁵ R⁹ MH+ 137 nd H PhO PhOH Me 551 138 nd Boc 4-t-Bu—PhO BnO H Me 721 139 nd H 4-t-Bu—PhO BnO H Me621 140 nd H (CF₃CO)₂N BnO H H 666 141 nd H PhCONH BnO H H 578 142 nd H4-pyridyl-CONH BnO H H 579 143 nd H (CF₃CO)₂N PhO H H 652 144 nd HPhCONH PhO H H 564 145 nd H 4-pyridyl-CONH PhO H H 565 146 nd H(CF₃CO)₂N MeO MeO H 620 147 nd H PhCONH MeO MeO H 532 148 nd H4-pyridyl-CONH MeO MeO H 533 149 nd H (CF₃CO)₂N H PhO H 652 150 nd HPhCONH H PhO H 564 151 nd H 4-pyridyl-CONH H PhO H 565 152 nd H PhCONH HBnO H 578 153 nd H 4-pyridyl-CONH H BnO H 579 154 nd H (CF₃CO)₂N H BnO H666 155 nd HO₂CCH₂CH₂CO 4-MeO—PhCONH PhO H H 694 156 nd HO₂CCH₂CH₂COPhCONH PhO H H 664 157 nd HO₂CCH₂CH₂CO 2-naphthyl- PhO H H 714 CONH 158nd HO₂CCH₂CH₂CO 4-Me—PhSO₂NH PhO H H 714 159 nd HO₂CCH₂CH₂CO4-MeO—PhCONH 2,3- H 652 benzo 160 nd HO₂CCH₂CH₂CO PhCONH 2,3- H 622benzo 161 nd HO₂CCH₂CH₂CO 2-naphthyl- 2,3- H 672 CONH benzo 162 ndHO₂CCH₂CH₂CO 4-Me—PhSO₂NH 2,3- H 672 benzo 163 nd HO₂CCH₂CH₂CO4-MeO—PhCONH H F H 620 164 nd HO₂CCH₂CH₂CO PhCONH H F H 590 165 ndHO₂CCH₂CH₂CO 2-naphthyl- H F H 640 CONH 166 nd HO₂CCH₂CH₂CO 4-Me—PhSO₂NHH F H 640 167 nd HO₂CCH₂CH₂CO 4-MeO—PhCONH BnO H H 708 168 ndHO₂CCH₂CH₂CO PhCONH BnO H H 678 169 nd HO₂CCH₂CH₂CO 2-naphthyl- BnO H H728 CONH 170 nd HO₂CCH₂CH₂CO 4-Me—PhSO₂NH BnO H H 728

TABLE 9

EPO/ EBP-Ig % inh @ 50 MS, cpd μM R^(a) R² R³ R⁴ R⁵ R⁹ MH+ 171 nb Cbz HH H H Me 527 172 15 Cbz H H H H H 513 173 5 Cbz H H H H t-Bu 569 174 23Cbz H MeO H MeO Me 587 175 1 Cbz 3,4- 3,4- Me 627 benzo benzo 176 −4 CbzPhO H PhO H Me 711 177 nd Cbz 2,3-benzo 2,3-benzo Me 627 178 36 Boc HNO₂ H NO₂ Me 583 179 30 Boc H NO₂ H NO₂ H 569 180 −4 Boc PhO H PhO H Me677 181 −9 Boc 4-t-Bu—PhO H 4-t-Bu—PhO H Me 790 182 18 H 4-t-Bu—PhO H4-t-Bu—PhO H Me 689 183 36 Boc NO₂ H NO₂ H Me 583 184 53 H NO₂ H NO₂ HMe 483 185 29 H NH₂ H NH₂ H Me 423 186 nd H n-Bu—CONH H n-Bu—CONH H Me591 187 nd H 2-furyl-CONH H 2-furyl-CONH H Me 611 188 nd H PhCONH HPhCONH H Me 631 189 nd H 4-Me—PhCONH H 4-Me—PhCONH H Me 659 190 nd H4-NO₂—PHCONH H 4-NO₂—PHCONH H Me 721 191 nd H 4-Me—PHSO₂NH H4-Me—PHSO₂NH H Me 731 192 nd H Cbz—NH H Cbz—NH H Me 691 193 nd H4-Br—PhCONH H 4-Br—PhCO H Me 789 194 nd H 2-MeO—PhCONH H 2-MeO—PhCONH HMe 691 195 nd H 3-MeO—PhCONH H 3-MeO—PhCONH H Me 691 196 nd H4-MeO—PhCONH H 4-MeO—PhCONH H Me 691 197 nd H CH₃CH═CHCONH HCH₃CH═CHCONH H Me 559 198 nd H C₂F₅CONH H C₂F₅CONH H Me 715 199 nd H2-naphthyl- H 2-naphthyl- H Me 731 CONH CONH 200 nd H EtO₂CCH₂CH₂CONH HEtO₂CCH₂CH₂CONH H Me 679 201 nd H CF₃CONH H CF₃CONH H Me 615 202 nd HMeSO₂NH H MeSO₂NH H Me 579

TABLE 10

EPO/ EBP-Ig % inh @ MS, cpd 50 μM R^(a) R² R³ R⁴ R⁵ Z MH+ 203 37 Boc H HH H 4-(MeCOCH₂CH₂)—PhNH 640 204 −6 H H H H H 4-(MeCOCH₂CH₂)—PhNH 540 20526 H H H H H n-Bu-NH 434 206 17 2-MeO—PhCO H H H H n-Bu—NH 568 207 204-MeO—PhCO H H H H n-Bu—NH 568 208 22 PhCO H H H H n-Bu—NH 538 209 252-MeO—PhCO H H H H n-Bu—NH 568 210 nd Boc H H H H 4-MeO—PhCH₂CH₂NH 612211 62 H H H H H 4-MeO—PhCH₂CH₂NH 512 212 −10 H H H H H n-Pr—NH 420 214nd Boc H H H H 3,4-di-MeO—PhCH₂CH₂NH 642 215 nd Boc H H H H3-MeO—PhCH₂CH₂NH 612 216 10 Boc H H H H 4-(PhCH═CHCH₂O)—PhCH₂NH 700 217nd Boc H H H H 4-HO—PhCH₂NH 584 218 nd Boc H H H H EtNH 506 219 nd Boc HH H H MeNH 492 220 45 H H H H H 4-(PhCH═CHCH₂O)—PhCH₂NH 600 221 48 H H HH H 3,4-di-MeO—PhCH₂CH₂NH 542 222 56 H H H H H 3-MeO—PhCH₂CH₂NH 512 223nd Boc H H H H 2-MeO—PhCH₂CH₂NH 612 224 51 H H H H H 2-MeO—PhCH₂CH₂NH512 225 10 Boc PhO H PhO H 4-MeO—PhCH₂CH₂NH 797 226 nd Boc H H H HPhCH₂CH₂NH 582 227 48 H H H H H PhCH₂CH₂NH 482 228 21 PhNHCO PhO H PhO H4-MeO—PhCH₂CH₂NH 816 229 22 4-PhO—PhNHCO H H H H 4-MeO—PhCH₂CH₂NH 723230 42 3,4-di-Cl—PhNHCO H H H H 4-MeO—PhCH₂CH₂NH 700 231 364-EtO2C—PhNHCO H H H H 4-MeO—PhCH₂CH₂NH 703 232 14 4-PhO—PhNHCO PhO HPhO H 4-MeO—PhCH₂CH₂NH 908 233 18 H H NO2 H NO2 4-MeO—PhCH₂CH₂NH 602 234nd Boc H H H H PhCH₂NH 568 235 49 H H H H H PhCH₂NH 468 236 nd Boc H PhH Ph 4-MeO—PhCH₂CH₂NH 765 237 55 HO₂CCH₂CH₂CO H H H H 3-MeO—PhCH₂CH₂NH612 238 39 H H Ph H Ph 4-MeO—PhCH₂CH₂NH 664 239 46 H PhO H PhO HPhCH₂CH₂NH 666 240 nd HO₂CCH₂CH₂CH₂CO PhO H PhO H PhCH₂CH₂NH 780 285 40H H H H H 4-(NH₂CO)piperidin-1-yl 489

TABLE 11

EPO/ EBP-Ig % inh @ MS, cpd 50 μM R^(a) R² R³ R⁴ R⁵ Z r [MH₂/2]+ 241 2t-Bu H BnO H BnO NH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 1 666 242 1 t-Bu H BnO H BnONH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NH 1 674 243 75 H H BnO H BnONH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 1 610 244 66 H H BnO H BnONH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NH 1 618 245 0 t-Bu H BnO H BnONH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 652 246 79 H H BnO H BnONH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 596 247 47 H n-Bu—CONH H PhO HNH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 575 248 56 H 2-furyl- H PhO HNH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 585 CONH 249 72 H 4-Me—PhCONH H PhO HNH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 609 250 78 H 4-Me—PhSO₂NH H PhO HNH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 645

TABLE 12

EPO/EBP-Ig % inh @ MS, cpd 50 μM R^(a) R² R⁴ Z r [MH₂/2]+ 251 49 H H HNH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 436 252 −4 t-Bu 4-t-Bu—PhO 4-t-Bu—PhONH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 1 803 253 −5 t-Bu 4-t-Bu—PhO 4-t-Bu—PhONH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NH 1 811 254 −9 t-Bu 4-t-Bu—PhO 4-t-Bu—PhONH(CH₂)₁₀NH 1 787 255 0 t-Bu 4-t-Bu—PhO 4-t-Bu—PhO NH(CH₂)₁₂NH 1 801 25610 t-Bu 4-t-Bu—PhO 4-t-Bu—PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 1 789

TABLE 13

EPO/EBP-Ig % inh @ MS, cpd 50 μM R^(a) Z [MH₂/2]+ 257 −26 BocNH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NH 731 258 −24 Boc NH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 723259 −13 Boc NH(CH₂)₁₂NH 721 260 −12 Boc NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 695 26151 H NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 595 262 93 HO₂CCH₂CH₂CONH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 695 263 88 HO₂C(CH₂)₃CONH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 709 264 89 HO₂CCH₂CMe₂CH₂CONH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 737 265 65 HO₂CCH₂CH₂CONH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 723 266 82 HO₂C(CH₂)₃CONH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 737 267 83 HO₂CCH₂CMe₂CH₂CONH(CH₂)₃O(CH₂)₄O(CH₂)₃NH 765 268 40 HO₂CCH₂CMe2CH₂CO NH(CH₂)₁₂NH 764 26955 HO₂CCH₂CH₂CH₂CO NH(CH₂)₁₂NH 735 270 56 HO₂CCH₂CH₂CO NH(CH₂)₁₂NH 721271 77 HO₂CCH₂CH₂CO NH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NH 731 272 78HO₂CCH₂CH₂CH₂CO NH(CH₂)₃O(CH₂CH₂O)₂(CH₂)₃NH 745

TABLE 14

EPO/ EBP-Ig % inh @ MS, cpd 50 μM R^(a) R² R⁴ Z n [MH₂/2]+ 273 ndHO₂CCH₂CH₂CO 4-Me—PhO 4-Me—PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 695 274 ndHO₂CCH₂CH₂CO PhO PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 667 275 nd HO₂CCH₂CH₂CO4-Me—PhO 4-Me—PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 727 276 nd HO₂CCH₂CH₂CO4-t-Bu—PhO 4-t-Bu—PhO NH(CH₂)₂O(CH₂)₂O(CH₂)₂NH 2 780 277 nd H PhO PhO(NHCH₂CH₂)₃N 3 813 278 nd H 4-Me—PhO 4-Me—PhO (NHCH₂CH₂)₃N 3 855 279 ndH 4-Me—PhO 4-Me—PhO (NHCH₂CH₂)₃N 3 903 280 nd HO₂CCH₂CH₂CO 4-Me—PhO4-Me—PhO (NHCH₂CH₂)₃N 3 1053 281 nd HO₂CCH₂CH₂CO 4-Me—PhO 4-Me—PhO(NHCH₂CH₂)₃N 3 1005 282 nd HO₂CCH₂CH₂CO PhO PhO (NHCH₂CH₂)₃N 3 963 283nd Boc PhO PhO NH(CH₂)₃NMe(CH₂)₃NH 2 666 284 nd Boc 4-Me—PhO 4-Me—PhONH(CH₂)₃NMe(CH₂)₃NH 2 694

TABLE 15

EPO/EBP-Ig % inh @ cpd 50 μM R¹ R² R³ MS, MH+ 285 −28 Me H H 473 286 46H BnO H 565 287 36 H 4-Me—PhO H 565 288 27 H 4-tBu—PhO H 607 289 20 H HPhO 551

TABLE 16A

EPO/EBP-Ig % inh @ cpd 50 μM R¹ R² R³ R⁴ 290 0 Me PhO H t-Bu 291 17 Me HPh t-Bu 292 11 Me 3-CF3—C6H4O H t-Bu 293 14 Me 3,4-Cl2—C6H3O H t-Bu 2949 Me 4-t-Bu—C6H4O H t-Bu 295 10 Me H Ph H 296 0 Me 3,4-Cl2—C6H3O H H 2970 Me 3-CF3—C6H4O H H 298 1 Me 4-t-Bu—C6H4O H H 299 nd Me PhO H H 300 57H PhO H H 301 25 H H Ph t-Bu 302 30 H 3,4-Cl2—C6H3O H t-Bu 303 21 H3-CF3—C6H4O H t-Bu 304 19 H 4-t-Bu—C6H4O H t-Bu 305 48 H H Ph H 306 21Me H H t-Bu 307 25 H 3,4-Cl2—C6H3O H H 308 25 H 3-CF3—C6H4O H H 309 13 H4-t-Bu—C6H4O H H 310 34 Me H H H

TABLE 16B MS, cpd MPLC solvent appearance empirical formula MH+ 29010-30% pale yellow oil C38H41NO5 592 EtOAc/hex 291 1:5 EtOAc/hex yellowoil C38H41NO3 560 292 1:5 EtOAc/hex yellow oil C40H39F6NO5 728 293 1:5EtOAc/hex yellow oil C38H37C14NO5 728 294 1:5 EtOAc/hex yellow oilC46H57NO5 704 295 off-white solid C34H33NO3/1 504 C2H4O2 296 amber solidC34H29C14NO5/1 672 C2H4O2 297 amber oil C36H31F6NO5/1 672 C2H4O2 298off-white solid C42H49NO5/1 648 C2H4O2 299 30% colorless oil C34H33NO5536 EtOAc/hex 300 sticky yellow solid C33H31NO5/1 522 C2H4O2 301 yellowsolid C37H39NO3 546 302 amber oil C37H35C14NO5 714 303 amber oilC39H37F6NO5 714 304 amber oil C45H55NO5 690 305 amber solid C33H31NO2/1490 C2HF3O2 306 light-yellow oil C26H33NO3/0.25 408 H2O 307 amber solidC33H27C14NO5/1 658 C2HF3O2 308 amber oil C35H29F6NO5/1 658 C2HF3O2 309off-white solid C41H47NO5/1 634 C2HF3O2 310 light yellow oil C22H25NO3/1352 C2H4O2

TABLE 17A

EPO/EBP-Ig % inh @ cpd 50 μM R¹ R² R³ 311 5.3 t-Bu PhO H 312 45 H PhO H

TABLE 17B cpd MPLC solvent appearance empirical formula MS, MH+ 311 10%EtOAc/hex pale yellow oil C36H37NO4 548 312 sticky brown C32H29NO4/1 492solid C2HF3O2

TABLE 18A

EPO/ EBP-Ig % inh @ cpd 50 μM R¹ R² R³ R⁴ 313 28 H H CF3(CH2)4NH(2-Cl—Cbz) 314 12 Me H CO2H (CH2)4NH2 315 nd Me H NO2(CH2)4NHBoc 316 20 Me OPh H (CH2)4NHBoc 317 13 Me 4- H (CH2)4NHBoct-Bu—C6H4O 318 14 Me H H (CH2)4NHCbz 319 nd Me H H (CH2)4NHCbz 320 17 MeH OMe (CH2)4NHCbz 321 42 Me CO2Me H (CH2)4NHCbz 322 nd Me H 2,3-(CH2)4NHCbz benzo 323 6 Me H CO2H (CH2)4NHCbz 324 nd Me H CO2Me nd

TABLE 18B cpd MPLC solvent appearance empirical formula MS, MH+ 313yellow oil C25H28ClF3N2O4\1 499 C2HF3O2 314 yellow oil C17H24N2O4 321315 30% EtOAc/hex dark yellow gum C21H31N3O6 422 316 20-50% EtOAc/hexpale yellow oil C27H36N2O5 469 317 pale yellow oil C31H44N2O5 525 318gum C24H30N2O4 411 319 pale yellow oil C24H30N2O4 411 320 2% MeOH/CH2Cl2yellow oil C25H32N2O5 441 321 yellow oil C26H32N2O6\1 469 C2H4O2 32225-50% EtOAc/hex clear residue C28H32N2O4 461 323 yellow oil C25H30N2O6455 324 yellow oil C26H32N2O6 469

TABLE 19A

EPO/EBP-Ig % inh @ cpd 50 μM R¹ R² R³ R⁴ 325 6 Me OPh H CH2CH2CO2H 326 0H OPh H CH2CH2CO2H 327 11 Me H Ph CH2CH2CO2H 328 33 Me 3,4-Cl2—C6H3O HCH2CH2CO2H 329 13 H H Ph CH2CH2CO2H 330 12 H 3-CF3—C6H4O H CH2CH2CO2H331 18 H 4-t-Bu—C6H4O H CH2CH2CO2H 332 17 H 3,4-Cl2—C6H3O H CH2CH2CO2H333 16 Me 3,4-benzo CH2CH2CO2-t-Bu 334 6 Me OPh H CH2CH2CO2-t-Bu 335 25Me H Ph CH2CH2CO2-t-Bu 336 32 Me 3,4-Cl2—C6H3O H CH2CH2CO2-t-Bu 337 0 HOPh H CH2CH2CO2-t-Bu 338 23 t-Bu 3-CF3—C6H4O H CH2CH2CO2-t-Bu 339 10t-Bu 4-t-Bu—C6H4O H CH2CH2CO2-t-Bu 340 14 H H Ph CH2CH2CO2-t-Bu 341 19 H3,4-Cl2—C6H3O H CH2CH2CO2-t-Bu

TABLE 19B cpd MPLC solvent appearance empirical formula MS, MH+ 325off-white solid C21H23NO5\1 370 C2F3HO2 326 fluffy white C20H21NO5\1 356solid C2HF3O2 327 off-white solid C21H23NO4\1 354 C2F3HO2 328 amber oilC21H21Cl2NO5\1 438 C2F3HO2 329 amber solid C20H21NO4\1 340 C2HF3O2 330amber oil C21H20F3NO5\1 424 C2HF3O2 331 amber oil C24H29NO5\1 412C2HF3O2 332 amber oil C20H19CL2NO5\1 424 C2HF3O2 333 10-25% EtOAc/hexyellow oil C23H29NO4 384 334 10-30% EtOAc/hex pale yellow oil C25H31NO5426 335 1:5 EtOAc/hex yellow oil C25H31NO4 410 336 1:5 EtOAc/hex yellowoil C25H29Cl2NO5 494 337 white powder C24H29NO5\0.4 412 H2O 338 1:5EtOAc/hex yellow oil C29H36F3NO5 536 339 1:5 EtOAc/hex yellow oilC32H45NO5 524 340 yellow solid C24H29NO4 396 341 white solidC24H27Cl2NO5 480

TABLE 20A

EPO/EBP-Ig % inh @ cpd 50 μM R¹ R² R³ R⁴ 342 0 Me H Ph CH2OH 343 37 Me4-t-Bu—C6H4O H CH2OH 344 4 Me 3-CF3—C6H4O H CH2OH 345 40 Me3,4-Cl2—C6H3O H CH2OH 346 28 Me OPh H CH2OH 347 23 H OPh H CH2OH 348 21H H Ph CH2OH 349 23 H 3,4-Cl2—C6H3O H CH2OH 350 23 H 3-CF3—C6H4O H CH2OH351 29 H 4-t-Bu—C6H4O H CH2OH 352 8 Me OPh H CH2O-t-Bu 353 24 Me H PhCH2O-t-Bu 354 31 Me 3,4-Cl2—C6H3O H CH2O-t-Bu 355 22 Me 3-CF3—C6H4O HCH2O-t-Bu 356 23 Me 4-t-Bu—C6H4O H CH2O-t-Bu 357 12 H 3-CF3—C6H4O HCH2O-t-Bu

TABLE 20B cpd MPLC solvent appearance empirical formula MS, MH+ 342off-white solid C19H21NO3\1 312 C2F3HO2 343 amber oil C23H29NO4\1 384C2F3HO2 344 amber oil C20H20F3NO4\1 396 C2F3HO2 345 amber oilC19H19Cl2NO4\1 396 C2F3HO2 346 EtOAc pale yellow oil C19H21NO4 328 347amber oil C18H19NO4\1 314 C2HF3O2 348 yellow solid C18H19NO3\1 298C2HF3O2 349 amber oil C18H17Cl2NO4\1 382 C2HF3O2 350 amber oilC19H18F3NO4\1 382 C2HF3O2 351 amber oil C22H27NO4\1 370 C2HF3O2 35210-30% EtOAc/hex pale yellow oil C23H29NO4 384 353 20% EtOAc/hexoff-white solid C23H29NO3 368 354 20% EtOAc/hex yellow oil C23H27Cl2NO4452 355 20% EtOAc/hex yellow oil C24H28F3NO4 452 356 20% EtOAc/hexyellow oil C27H37NO4 440 357 white solid C23H26F3NO4 438

TABLE 21A

EPO/EBP-Ig % inh @ cpd 50 μM R¹ R² R³ R⁴ 358 0 H H CF3 (s)-CH(OH)CH3 35925 Me CO2Me H (s)-CH(OMe)CH3 360 18 Me H H Bn 361 24 Me CO2Me H Bn 362 0H H CF3 CH2(4-HOC6H4) 363 33 Me CO2Me H CH2(4-MeOC6H4) 364 16 Me H HCH2(indol-3-yl) 365 0 H H CF3 CH2CH2SMe 366 38 Me CO2Me H CH2CO2Me 367 0H H CF3 CH2CONH2 368 40 Me CO2Me H CH2SBn 369 12 H H CF3 i-Bu 370 0 H HCF3 i-Pr 371 16 Me CO2Me H i-Pr 372 0 Me H H Me

TABLE 21B cpd MPLC solvent appearance empirical formula MS, MH+ 358amber oil C14H16F3NO3\1 304 C2HF3O2 359 amber oil C17H23NO5\1 322 C2H4O2360 20% EtOAc/hex light-yellow C19H21NO2 296 oil 361 amber oilC22H25NO5\1 354 C2H4O2 362 amber oil C19H18F3NO3\1 366 C2HF3O2 363 amberoil C22H25NO5\1 384 C2H4O2 364  1:2 EtOAc/hex tan solid C21H22N2O2 335365 amber oil C15H18F3NO2S\1 334 C2HF3O2 366 amber oil C17H21NO6\1 336C2H4O2 367 amber oil C14H15F3N2O3\1 317 C2HF3O2 368 amber oilC22H25NO4S\1 400 C2H4O2 369 amber oil C17H22F3NO2\1 316 C2HF3O2 370amber oil C15H18F3NO2\1 302 C2HF3O2 371 amber oil C17H23NO4\1 306 C2H4O2372 20% EtOAc/hex yellow oil C13H17NO2\0.10 220 C4H8O2

1. A pharmaceutical composition comprising a compound of the formula:

wherein: R¹ is H or C₁₋₅alkyl; R² is H, phenoxy, benzyl, substitutedphenoxy (where the substituents are selected from C₁₋₅alkyl, C₁₋₅alkoxy,hydroxy, halo, trifluoromethyl, nitro, cyano, and amino), or CO₂Me; R³is H, phenyl, triflouromethyl; and R⁴ is the side chain of a natural orunnatural α-amino acid, where if said side chain contains a protectablegroup, that group may be protected with a member of the group consistingof succinyl, glutaryl, 3,3-dimethylglutaryl, C₁₋₅alkyl,C₁₋₅alkoxycarbonyl, acetyl, N-(9-flourenylmethoxycarbonyl),trifluoroacetyl, omega-carboxyC₁₋₅alkylcarbonyl, t-butoxycarbonyl,benzyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, phenylsulfonyl,ureido, t-butyl, cinnamoyl, trityl, 4-methyltrityl,1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl, tosyl,4-methoxy-2,3,6-trimethylbenzenesulfonyl, phenylureido, and substitutedphenylureido(where the phenyl substituents are phenoxy, halo,C₁₋₅alkoxycarbonyl).
 2. A pharmaceutical composition comprising anactive drug component and the compound of claim
 1. 3. The composition ofclaim 2 wherein said active drug component is combined with an oral,non-toxic pharmaceutically acceptable inert carrier.
 4. The compositionof claim 3 wherein said carrier is ethanol, glycerol, or water.
 5. Thecomposition of claim 2 further comprising binders, lubricants,disintegrating agents, or coloring agents.
 6. The composition of claim 5wherein said binders are selected from the group consisting of starch,gelatin, natural sugars, corn sweeteners, natural and synthetic gumscarboxymethylcellulose, polyethylene glycol, and waxes.
 7. Thecomposition of claim 5 wherein said lubricants are selected from thegroup consisting of sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, and sodium chloride.
 8. The compositionof claim 5 wherein said disintegrating agents are selected from thegroup consisting of starch, methyl cellulose, agar, bentonite, andxanthan gum.
 9. The composition of claim 2 contained in a topicaladministration.
 10. The composition of claim 9 wherein said active drugcomponent can be admixed with carrier materials selected from the groupconsisting of alcohols, aloe vera gel, allontoin, glycerine, vitamin Aand E oils, mineral oil, PPG2 myristyl propionate.
 11. An oralcomposition comprising an EPO receptor modulating compound comprisingthe compound of claim 1 having an active drug component being combinedwith an oral, non-toxic pharmaceutically acceptable inert carrier. 12.The composition of claim 11 wherein said carrier is ethanol, glycerol,or water.
 13. The composition of claim 11 further comprising binders,lubricants, disintegrating agents, or coloring agents.
 14. Thecomposition of claim 13 wherein said binders are selected from the groupconsisting of starch, gelatin, natural sugars, corn sweeteners, naturaland synthetic gums carboxymethylcellulose, polyethylene glycol, andwaxes.
 15. The composition of claim 13 wherein said lubricants areselected from the group consisting of sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, and sodiumchloride.
 16. The composition of claim 13 wherein said disintegratingagents are selected from the group consisting of starch, methylcellulose, agar, bentonite, and xanthan gum.